A novel and simple colorimetric assay for human serum lipase.

A new and simple colorimetric method for human serum lipase [EC 3.1.1.3] assay has been developed, using 2,3-dimercaptopropan-1-ol tributyroate as a substrate, 5,5'-dithiobis(2-nitro-benzoic acid) as a chromogenic reagent, phenylmethylsulfonyl fluoride as an inhibitor of serum esterases, and sodium dodecylsulfate as a lipase activator. The method requires only 50 micron1X2 of serum sample and a reaction time of less than 30 min. The method is reproducible and sensitive enough to measure low levels of lipase activity in normal and abnormal sera. The gel filtration of serum samples on a Sephadex G-200 column gave one peak of lipase activity, when measured by the present method, and the molecular weight of the enzyme was identical with that of lipase of human pancreatic origin, confirming the specificity of this new method for the serum lipase.     

A simple method for isolating trypsin from trichloroacetic acid extracts of bovine pancreas

Trichloroacetic acid was used to isolate trypsin and trypsinogen from bovine pancreas. Trypsinogen, which is at first soluble in trichloroacetic acid, slowly forms a sediment. In alkaline medium and in the presence of calcium chloride, proenzyme is activated to enzyme which has high proteolytic, esterase, and amidase activity and is homogenous in polyacrylamide gel electrophoresis. It is suggested that the gradual reduction of trypsinogen solubility in trichloroacetic acid is associated with the presence of basic pancreatic trypsin inhibitor in trichloroacetic acid extracts from a bovine pancreas.     

A rapid and sensitive lipase assay using triethylaminoethyl cellulose columns

A procedure has been developed for the separation of labeled fatty acids from tri-, di-, and monoglycerides using small disposable columns of TEAE-cellulose. This procedure is used as the basis of a lipase assay which is rapid, sensitive and unaffected by wide variations in the composition of the reaction mixtures. 0.5 nmole [¹⁴C]oleic acid can be detected, and the entire procedure requires less than 3 min.     

Determination of lipoprotein lipase activity using a novel fluorescent lipase assay

A novel, real-time, homogeneous fluorogenic lipoprotein lipase (LPL) assay was developed using a commercially available substrate, the EnzChek lipase substrate, which is solubilized in Zwittergent. The triglyceride analog substrate does not fluoresce, owing to apposition of fluorescent and fluorescent quenching groups at the sn-1 and sn-2 positions, respectively, fluorescence becoming unquenched upon release of the sn-1 BODIPY FA derivative following hydrolysis. Increase in fluorescence intensity at 37°C was proportional to LPL concentration. The assay was more sensitive than a similar assay using 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6-methylresorufin ester) and was validated in biological samples, including determination of LPL-specific activity in postheparin mouse plasma. The simplicity and reproducibility of the assay make it ideal for in vitro, high-throughput screening for inhibitors and activators of LPL, thus expediting discovery of drugs of potential clinical value.     

A simple competitive enzyme-linked immunosorbent assay using antigen-beta-galactosidase fusions

The fusion of the N-terminal 461 bp of the human interferon-alpha 2 (INF) in frame to the beta-galactosidase gene from Escherichia coli is described. The presence of the expected DNA sequence was shown by restriction mapping and DNA sequencing. A fusion protein was demonstrated in crude extracts of E. coli by Western blots using polyclonal anti-beta-galactosidase and monoclonal anti-IFN antibodies. Using monoclonal antibodies specific for the N-terminal region of IFN-alpha and cell-free extracts from an E. coli strain containing the fusion protein, we set up a simple competitive enzyme-linked immunosorbent assay for human interferon. The test described here was linear down to a lower detection limit of at least 1000 Units, or 5 ng human IFN.     

Development of a trichloroacetic acid precipitation assay for covalent adducts of thymidylate synthase

The use of trichloroacetic acid as a protein precipitant and denaturant in the quantitative measurement of covalent complexes of thymidylate synthase is described. Enzyme inactivated with N[3H]ethylmaleimide and inhibitory ternary complex (formed with native enzyme, 5-[6-3H]fluoro-2'-deoxyuridylate, and methylenetetrahydrofolate) served as reagents which were used to establish the conditions under which trichloroacetic acid precipitation, washing, and solubilization steps provided quantitative results. The ternary complex formed by dihydrofolate reductase with [3H]methotrexate and NADPH was used as a control to assess whether tight, but noncovalent, enzyme:ligand complexes survived trichloroacetic acid precipitation. The fact that no counts above background were detected in the pellet of precipitated protein demonstrated that the noncovalent complexes were completely dissociated by this treatment. The dynamic range of linear response for the inhibitory ternary complex of thymidylate synthase spanned five orders of magnitude, and the assay detected levels of enzyme as low as 10 fmol, a value which was essentially limited by the specific radioactivity of 5-[6-3H]fluoro-2'-deoxyuridylate. The ability of the enzyme to bind 5-[6-3H]fluoro-2'-deoxyuridylate specifically, as measured by the trichloroacetic acid assay, generated a specific binding value of 13.4 nmol of enzyme/mg protein (assuming a binding ratio of 1.5 for the inhibitory ternary complex). Specific binding values were compared to specific activity values (obtained from the spectrophotometric assay) at each stage of purification of the enzyme from Lactobacillus casei and were found to give parallel results. The characteristics of the trichloracetic acid assay procedure, which exclusively detects covalent enzyme-ligand adducts, are compared to those for other ligand binding assays for thymidylate synthase.     

A rapid assay for lipoprotein lipase

A rapid assay for lipoprotein lipase activity employing a (14)C-labeled substrate is described. The method is very sensitive and suitable for routine use.     

A simple vertebrate collagenase assay using soluble radioactive collagen substrate

A highly sensitive assay for vertebrate collagenase has been developed using [14C]proline- or [3H]proline-labeled collagen as soluble substrate. The substrate was easy to prepare, gave high specific activity (1.4 X 10(6) cpm/mg collagen), and was stable at -20 degrees C for a long period. The digestion reaction for the assay was done at 21 degrees C to minimize the cleavage of collagen by proteases other than collagenase and to protect the 3/4 and 1/4 cleavage fragments of collagen from being further attacked by proteases. The cleaved products were denatured and then separated from undigested native collagen by precipitation with 1 M NaCl at pH 3.5. The conditions selected for denaturation and separation gave better discrimination between the cleaved products and uncleaved substrate than did conditions used in some other assays. The digestion products can be examined further by gel electrophoresis at the end of the assay to confirm the activity of vertebrate collagenase. This assay can also be adapted to assess telopeptidase activity independently of collagenase activity.     

Fatty acid esterification using nylon-immobilized lipase

The esterification of a long-chain fatty acid was conducted using a nylon-immobilized lipase from Candida cylindracea in a nearly anhydrous, nonpolar organic medium, hexane. Butyl laurate was produced from lauric acid and n-butanol at a maximum initial reaction rate of 37 mmol/h. g immobilized enzyme when the substrates were present in equimolar amounts at an initial concentration of 0.5 mol/L. Lower rates were obtained using nonstoichiometric amounts of the substrates. The rate of reaction increased with temperature, reaching a maximum between 35 and 45 degrees C and decreasing sharply at higher temperatures. (c) 1995 John Wiley & Sons, Inc.     

A simple assay for toxic chemicals using a bacterial indicator

A simple, inexpensive and rapid technique to measure toxicity has been developed using Rhizobium meliloti as the indicator organism and its rapid reduction of the tetrazolium dye MTT (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide). Toxic chemicals inhibited the reduction in this bacterium but not in others. Nearly 50 organic chemicals and 14 minerals were tested: inhibition was proportional to the concentration of the toxic chemical. The mechanism to account for both the reduction of the dye and the inhibition of reduction is uncertain. This method provides a simple and inexpensive way to determine the critical concentration of toxic compounds. The assay provides values comparable to those provided by the fat head minnow (Pimephales promelas). Results are also comparable to those obtained with the Microtox and Polytox assays, two commercial assays that use bacteria as indicator organisms.     

A simple and highly sensitive radioenzymatic assay for lysophosphatidic acid quantification

The objective of the present work was to develop a simple and sensitive radioenzymatic assay to quantify lysophosphatidic acid (LPA). For that, a recombinant rat LPA acid acyltransferase (LPAAT) produced in Escherichia coli was used. In the presence of [(14)C]oleoyl-CoA, LPAAT selectively catalyzes the transformation of LPA and alkyl-LPA into [(14)C]phosphatidic acid. Acylation of LPA was complete and linear from 0 to 200 pmol with a minimal detection of 0.2 pmol. This method was used to quantify LPA in butanol-extracted lipids from bovine sera, as well as from human and mouse plasma.This radioenzymatic assay represents a new, simple, and highly sensitive method to quantify LPA in various biological fluids.     

A simple and sensitive assay for 1-aminocyclopropane-1-carboxylic acid

A simple, rapid, and sensitive method for the quantitative determination of 1-amino-cyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene in plant tissues, is described. The assay is based on the liberation of ethylene from ACC with NaOCl in the presence of Hg²⁺; ethylene is assayed by gas chromatography. The yield is normally 80% and can be determined by internal standards. The method is quite specific and can detect as little as 5 pmol of ACC.     

Microbial dehalogenation of trichloroacetic acid

A pure bacterial culture and a two-membered mixed culture were isolated that degraded trichloroacetic acid if a second, readily metabolizable substrate was present in the growth medium. Previous doubts over the microbial dehalogenation of trichloroacetic acid (TCA) may be due to its inability to act as a sole carbon and energy source. TCA dehalogenation was associated with conventional 2-haloalkanoic acid dehalogenases but oxalate, the putative dehalogenase product, was not detected. CO₂ was produced rapidly and concomitantly with Cl^– ion release during dehalogenation of TCA. An alternative mechanism is suggested for TCA dehalogenation via an initial decarboxylation reaction. This mechanism predicts that carbon monoxide is a product of TCA decarboxylation and it was significant that one of the organisms isolated,Pseudomonas carboxydohydrogens, was a carboxytroph and a second was an unidentified facultative methylotroph.     

A simple and sensitive assay for ascorbate using a plate reader

We have developed a rapid, inexpensive, and reliable assay for the determination of ascorbate using a plate reader. In this assay, ascorbic acid is oxidized to dehydroascorbic acid using Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy) and then reacted with o-phenylenediamine to form the condensation product, 3-(dihydroxyethyl)furo[3,4-b]quinoxaline-1-one. The rate of appearance of this product is monitored over time using fluorescence. With this method, it is possible to analyze 96 wells in less than 10min. This permits the analysis of 20 samples with a full set of standards and blanks, all in triplicate. The assay is robust for a variety of samples, including orange juice, swine plasma, dog plasma, and cultured cells. To demonstrate the usefulness of the assay for the rapid determination of experimental parameters, we investigated the uptake of ascorbate and two different ascorbate derivatives in U937 cells. We found similar plateau levels of intracellular ascorbate at 24h for ascorbate and ascorbate phosphate. However, the intracellular accumulation of ascorbate via the phosphate ester had an initial rate that was three to five times slower than that via the palmitate ester. Only lower concentrations of the palmitate ester could be examined because the ethanol needed as solvent decreased cell viability; it behaved similarly to the other two compounds at lower concentrations. To come to these conclusions, only nine plates needed to be analyzed to provide us with the end result after only 7h of analysis. This clearly demonstrates the strength of the plate reader assay, which allows the analysis of large-sample sets in a fraction of the time required for the methods that are most commonly used today. The assay is quick, is very economical, and provides results with uncertainties on the order of only 5%.     

A simple,real-time assay of horseradish peroxidase using biolayer interferometry

ABSTRACT

Horseradish peroxidase (HRP) isoenzyme C1a is one of the most widely used enzymes for various analytical methods in bioscience research and medical fields. In these fields, real-time monitoring of HRP activity is highly desirable because the utility of HRP as a reporter enzyme would be expanded. In this study, we developed a simple assay system enabling real-time monitoring of HRP activity by using biolayer interferometry (BLI). The HRP activity was quantitatively detected on a BLI sensor chip by tracing a binding response of tyramide, a substrate of HRP, onto an immobilized protein. This system could be applied to analyses related to oxidase activity, as well as to the functional analysis of recombinant HRP.     

A simple bioassay for abscisic acid using cucumber hypocotyls

A simple bioassay based on the inhibition by abscisic acid (ABA) of cucumber (Cucumis sativus L., cv. National Pickling) hypocotyl elongation was developed. Sections of 3-day-old dark-grown cucumber hypocotyl taken from 0–5 mm immediately below the cotyledon were used for the assay. A dark incubation period of 20 h was followed by an exposure to light for 24 h. Under these conditions, the inhibition of hypocotyl elongation is proportional to the abscisic acid applied. The minimum detectable level of abscisic acid was 10^–9 M, and the range of linear response to abscisic acid was between 10^–7 and 10^–3 M. This assay is 10 times more sensitive than the cucumber cotyledon greening bioassay for abscisic acid.