Fixed-time Assay Research Articles

Electrochemical flow injection approach for routine screening of lipase activity in pancreatic preparations

A state-of-the-art strategy for the determination of lipase activity in pancreatic preparations using flow injection analysis (FIA) with electrochemical detection (FIA-ED) is described. The procedure is based on the enzymatic reaction of a specific substrate (1,3-dilinoleoyl-glycerol) with lipase from porcine pancreas and determination of enzymatically formed linoleic acid (LA) at +0.4 V by applying a cobalt (II) phthalocyanine-multiwalled carbon-nanotubes modified carbon paste electrode (Co(II)PC/MWCNT/CPE). In order to get a high-performance analytical method, sample preparation, flow system, and electrochemical conditions were optimized. Under optimized conditions the lipase activity of porcine pancreatic lipase was calculated to be 0.47 units per mg lipase protein based on the definition that 1 unit hydrolyses 1 microequivalent linoleic acid from 1,3-dilinoleoyl-glycerol per 1 min at pH 9 and 20 °C (kinetic measurement: 0–25 min). Moreover, the developed procedure was shown to be easily adaptable for the fixed-time assay (incubation time 25 min) as well. In this case, linear correlation between flow signal and lipase activity was found in the range from 0.8 to 18 U L−1. LOD and LOQ were determined to be 0.3 U L−1 and 1 U L−1, respectively. The kinetic assay was further preferred for the determination of lipase activity in commercially available pancreatic preparations. The lipase activities of all preparations obtained by the present method were found to be in good correlation with those obtained by the titrimetric method and declared by manufacturers.

Quantitation of Glutamine Synthetase 1 Activity in Drosophila melanogaster.

Protocols to assay the activity of glutamine synthetase (GS) are presented as they have been used in our laboratory to correlate the expression levels of the gene encoding Drosophila GS1 gene, the GS1 protein levels, and its activity in extracts of larvae and heads from Drosophila melanogaster. The assays are based on the glutamine synthetase-catalyzed formation of γ-glutamylhydroxylamine in the presence of ATP, L-glutamate, and hydroxylamine, in which hydroxylamine substitutes for ammonia in the reaction. Formation of γ-glutamylhydroxylamine is monitored spectrophotometrically in discontinuous assays upon complex formation with FeCl3. Fixed-time assays and those based on monitoring the time-course of product formation at different reaction times are described. The protocols can be adapted to quantify glutamine synthetase activity on biological materials other than Drosophila.

239 Increasing Dietary Soybean Meal Level Improves Growth and Feed Conversion Efficiency in Healthy Pigs and Reduces GHG Emissions

Abstract The objective of this study was to determine the growth response to increasing soybean meal (SBM) dose (low, medium, high) compared to a high-energy, low SBM reference diet (HE L-SBM). A total of 2283 PIC terminal pigs (initial BW=44.25+1.14 kg) were used in a fixed time assay (ca. 133.63+1.23 kg). Pigs were placed in 96 mixed-sex pens (23-25 pigs/pen; 0.75 m2/pig), blocked by pen BW and allotted to 4 diets (HE L-SBM, L-SBM, M-SBM, H-SBM), which were fed in 4 phases beginning at 40.8, 59.0, 86.2, 104.4 kg BW. Dietary level of SBM corresponded to 19.0, 19.0, 24.5, and 30.0% respectively (phase 1); 14.0, 14.0, 18.0, and 22.0% (phase 2); 9.5, 9.5, 13.0, and 16.5% (phase 3) and 7.5, 7.5, 10.5, and 13.0% (phase 4). The HE L-SBM diet contained 20% DDGS, SBM equal to L-SBM diet, amino acids, 3.0% CWG. Diets exceeded NRC (2012) recommendations for nutrients, were iso-nutrient and SID Lysine:NE ratio was kept constant within dietary phase. The HE L-SBM diet had ca. 52 kcal NE/kg advantage to L, M, H-SBM diets (isocaloric). Early growth (phase 1) tended to be greatest for H-SBM (0.917, 0.919, 0.938 kg/d, P=0.118); each exceeding the summer reference diet (HE L-SBM 0.883, P< 0.001). In phase 2, pigs fed SBM diets grew similarly at each level (0.902, 0.920, 0.917 kg/d); HE L-SBM was inferior (0.883, P< 0.010). All diets performed similar for growth in phases 3, 4 (0.932, 0.736 kg/d). Carcass weight was the greatest for M-SBM, L-SBM diets (98.5, 98.6 kg) compared to HE L-SBM (97.1, P< 0.072). Whole-body G:F was greater for H-SBM vs L-SBM, M-SBM (0.354, 0.346, 0.339; P< 0.116, P< 0.005) and equal to HE L-SBM (0.355). Thus, early growth benefitted from H-SBM; DDGS reduced gain despite fat addition. Caloric efficiency favored H-SBM (9.466 Mcal/kg gain) over HE L-SBM (10.098), as did predicted GHG emission (< 4.6%).

A high-throughput screening assay for pyruvate carboxylase

Pyruvate carboxylase (PC) catalyzes the conversion of pyruvate to oxaloacetate (OAA), an important metabolic reaction in a wide range of organisms. Small molecules directed against PC would enable detailed studies on the metabolic role of this enzyme and would have the potential to be developed into pharmacological agents. Currently, specific and potent small molecule regulators of PC are unavailable. To assist in efforts to find, develop, and characterize small molecule effectors of PC, a novel fixed-time assay has been developed based on the reaction of OAA with the diazonium salt, Fast Violet B (FVB), which produces a colored adduct with an absorbance maximum at 530 nm. This fixed time assay is reproducible, sensitive and responsive to known effectors of Rhizobium etli PC, Staphylococcus aureus PC, and Listeria monocytogenes PC, and is highly amenable to high-throughput screening. The assay was validated using a plate uniformity assessment test and a pilot screen of a library of 1280 compounds. The results indicate that the assay is suitable for screening small molecule libraries to find novel small molecule effectors of PC.

Discovery of Novel Small Molecule Inhibitors of Bacterial Pyruvate Carboxylase

Staphylococcus aureus and Listeria monocytogenes are pathogenic bacteria that can cause serious infections for immunocompromised individuals1, 2. Bacterial metabolism has been linked to pathogenicity and, in particular, the metabolic enzyme pyruvate carboxylase (PC) has been shown to be an important contributor to L. monocytogenes virulence1, 2. Small molecules that could inhibit bacterial PC activity would better reveal the role of this enzyme in a wide range of bacterial infections and could have the potential to be developed into antimicrobial agents. However, there are currently no known specific or potent small molecule inhibitors of PC activity3. We aim to find, develop, and characterize novel small molecule inhibitors of PC that will aid in understanding the structure, mechanism, and regulation of this enzyme. PC catalyzes the conversion of pyruvate to oxaloacetate (OAA) and a novel fixed‐time assay has been developed to detect OAA with a diazonium salt, Fast Violet B (FVB). The validated assay has been used to screen specific subsets of rationally designed inhibitors as well as compounds in an unbiased high‐throughput screen. Using both approaches, we have discovered novel small molecule inhibitors of bacterial PC that have the potential to be developed into valuable research tools and future antimicrobial agents.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A continuous assay for l-talarate/galactarate dehydratase using circular dichroism

l-Talarate/galactarate dehydratase (TGD) is a member of the enolase superfamily of enzymes and catalyzes the dehydration of either meso-galactarate or l-talarate to form 5-keto-4-deoxy-d-glucarate (5-KDG). To facilitate study of this enzyme and other galactarate dehydratases, a continuous circular dichroism-based assay has been developed. Using recombinant enzyme from Salmonella typhimurium (StTGD), the rates of StTGD-catalyzed conversion of m-galactarate to 5-KDG were determined by following the change in ellipticity at 323 nm. The apparent molar ellipticity ([θ]323) for the 5-KDG formed was determined to be 202 ± 2 deg cm2 dmol−1, which was used to convert observed rates (Δθ/Δt) into concentration-dependent rates (Δc/Δt). The kinetic parameters Km, kcat, and kcat/Km were 0.38 ± 0.05 mM, 4.8 ± 0.1 s−1, and 1.3 (±0.2) × 104 M−1s−1, respectively. These values are in excellent agreement with those published previously [Yew, W.S. et al. (2007) Biochemistry46, 9564–9577] using a coupled assay system. To demonstrate the utility of the assay, the inhibition constant (Ki = 10.7 ± 0.4 mM) was determined for the competitive inhibitor tartronate. The continuous CD-based assay offers a practical and efficient alternative method to the coupled assay that requires access to 5-KDG aldolase, and to the labor-intensive, fixed-time assays.

Evaluation of peroxidase assay and effect of thermal blanching on sapota and fig fruits

Abstract A spectrophotometric assay was developed for the quantification of peroxidase activity in the presence of para -phenylenediamine dihydrochloride (PPDD) and 8-hydroxyquinolene (8-HQ) as chromogenic co-substrates in KH 2 PO 4 /NaOH buffer solution at pH 7.8. The resulting violet coloured product had maximum absorbance at 540 nm. Under optimized condition, the linearity range of hydrogen peroxide (H 2 O 2 ) was 1–124 µM and that of peroxidase was 0.2958–14 nM and 0.0739–2.3674 nM by kinetic and fixed time assays, respectively. The Michaelis–Menten ( K m ) constant, catalytic efficiency and catalytic power were 38.72 µM, 0.2613 × 10 6 M −1 min −1 and 2.4748 × 10 −3 min −1 , respectively. The obtained broad and sensitive linearity range of H 2 O 2 and peroxidase assays and lower value of Michaelis–Menten constant indicate the superiority of this method. The developed method was successfully applied to study the effect of blanching treatment of peroxidase in sapota and fig fruit extracts and this is helpful in fixing blanching temperature and blanching time for fruits.

Spectrophotometric assay of creatinine in human serum sample

A new spectrophotometric method for the analysis of creatinine concentration in human serum samples is developed. The method explores the oxidation of p-methylamino phenol sulfate (Metol) in the presence of copper sulfate and creatinine which yields an intense violet colored species with maximum absorbance at 530nm. The calibration graph of creatinine by fixed time assay ranged from 4.4 to 620μM. Recovery of creatinine in human serum samples varied from 101% to 106%. Limit of detection and limit of quantification were 0.145μM and 0.487μM respectively. Sandell’s sensitivity was 0.112μgcm−2 and molar absorptivity was 0.101×104Lmol−1cm−1. Within day precision was 2.5–4.8% and day-to-day precision range was 3.2–7.8%. The robustness and ruggedness of the method expressed in RSD values ranged from 0.78% to 2.12% and 1.32% to 3.46% respectively, suggesting that the developed method was rugged. This method provides good sensitivity and is comparable to standard Jaffe’s method with comparatively less interference from foreign substances.

Engineered antifouling microtopographies: kinetic analysis of the attachment of zoospores of the green alga Ulva to silicone elastomers

Microtopography has been demonstrated as an effective deterrent to biofouling. The majority of published studies are fixed-time assays that raise questions regarding the kinetics of the attachment process. This study investigated the time-dependent attachment density of zoospores of Ulva, in a laboratory assay, on a micropatterned and smooth silicone elastomer. The attachment density of zoospores was reduced on average 70–80% by the microtopography relative to smooth surfaces over a 4 h exposure. Mapping the zoospore locations on the topography revealed that they settled preferentially in specific, recessed areas of the pattern. The kinetic data fit, with high correlation (r 2 > 0.9), models commonly used to describe the adhesion of bacteria to surfaces. The grouping of spores on the microtopography indicated that the pattern inhibited the ability of attached spores to recruit neighbors. This study demonstrates that the antifouling mechanism of topographies may involve disruption of the cooperative effects exhibited by fouling organisms such as Ulva.

Fluorimetric determination of ampicillin by use of non-toxic catalysts. Estimation of beta-lactamase activity and parameters.

A fluorescence assay based on the use of biological reducing agents as catalysts rather than heavy metal ions has been developed for estimation of ampicillin concentrations. The assay is based on the conversion of ampicillin to its penicilloate, by treatment with sodium hydroxide, then neutralization, dilution with 0.5 M acetate buffer at pH4 and heating for 30 min at 100 degrees C in the presence of ascorbic acid (0.5 mg) and EDTA (50 microM). Reduced glutathione, cysteine and N-acetylcysteine also catalysed the development of fluorescence. A practical sensitivity range of 0.5-50 microM ampicillin was used. The assay was used to estimate ampicillin in some biological solutions to which the antibiotic has been added. Milk, blood serum, trypticase soy broth and nutrient broth containing 25 microM antibiotic assayed at 18.5, 21.7, 16.5 and 14.7 microM, respectively, with standard deviations between 1.2 and 0.7%. The low results were attributed to binding of some ampicillin by proteins or peptides which were removed by pretreatment. Urine containing 5 mM ampicillin assayed at 4.97 mM with a standard deviation of 3%. A modification of the procedure was used to measure beta-lactamase activity against ampicillin in several organisms in a fixed time assay. Kinetic parameters of a commercial beta-lactamase preparation from Bacillus cereus could also be determined by an additional modification. In both instances a correction was required for the intrinsic fluorescence of ampicillin remaining in the solution. The preparation examined had a Michaelis constant (Km) of 0.32 mM, a maximum velocity (Vmax) of 5398 micromol ampicillin hydrolysed mg(-1) min(-1), an apparent catalytic constant (Kcat, turnover number) of 20,220 s(-1) and a Kcat/Km ratio of 0.63 x 10(7) M(-1) s(-1). The major advantage of using this assay technique is that toxic metals are not used in the development of fluorescence so it is more environmentally acceptable. The technique is useful for examining beta-lactamase activity against ampicillin and might be useful for pharmaceutical products-for determining available therapeutic levels and for monitoring the activity of penicillin acylase against the penicilloate of ampicillin.

A continuous assay for α-methylacyl-coenzyme A racemase using circular dichroism

α-Methylacyl-coenzyme A racemase (AMACR) catalyzes the epimerization of (2 R)- and (2 S)-methyl branched fatty acyl-coenzyme A (CoA) thioesters. AMACR is a biomarker for prostate cancer and a putative target for the development of therapeutic agents directed against the disease. To facilitate development of AMACR inhibitors, a continuous circular dichroism (CD)-based assay has been developed. The open reading frame encoding AMACR from Mycobacterium tuberculosis (MCR) was subcloned into a pET15b vector, and the enzyme was overexpressed and purified using metal ion affinity chromatography. The rates of MCR-catalyzed epimerization of either (2 R)- or (2 S)-ibuprofenoyl-CoA were determined by following the change in ellipticity at 279 nm in the presence of octyl-β- d-glucopyranoside (0.2%). MCR exhibited slightly higher affinity for (2 R)-ibuprofenoyl-CoA ( K m = 48 ± 5 μM, k cat = 291 ± 30 s −1), but turned over (2 S)-ibuprofenoyl-CoA ( K m = 86 ± 6 μM, k cat = 450 ± 14 s −1) slightly faster. MCR expressed as a fusion protein bearing an N-terminal His 6-tag had a catalytic efficiency ( k cat/ K m) that was reduced 22% and 47% in the 2 S → 2 R and 2 R → 2 S directions, respectively, relative to untagged enzyme. The continuous CD-based assay offers an economical and efficient alternative method to the labor-intensive, fixed-time assays currently used to measure AMACR activity.

DNA Binding and Cleavage by the Fowlpox Virus Resolvase

The first steps of poxvirus DNA synthesis yield concatemeric arrays of covalently linked genomes. The virus-encoded Holliday junction resolvase is required to process concatemers into unit-length genomes for packaging. Previous studies of the vaccinia virus resolvase have been problematic due to poor protein solubility. We found that fowlpox virus resolvase was much more tractable. Fowlpox resolvase formed complexes with a variety of branched DNA substrates, but not linear DNA, and had the highest affinity for a Holliday junction substrate, illustrating a previously unappreciated affinity for Holliday junctions over other substrates. The cleavage activity was monitored in fixed time assays, showing that, as with vaccinia resolvase, the fowlpox enzyme could cleave a wide array of branched DNA substrates. Single turnover kinetic analysis revealed the Holliday junction substrate was cleaved 90-fold faster than a splayed duplex substrate containing a single to double strand transition. Multiple turnover kinetic analysis, however, showed that the cleavage step was not limiting for the full reaction cycle. Cleavage by resolvase was also tightly coupled at symmetrical positions across the junction, and coupling required the complete Holliday junction structure. Last, we found that cleavage of an extruded cruciform yielded a product, which after treatment with ligase, had the properties expected for covalently closed DNA hairpin ends, as is seen for poxvirus genome monomers. These findings provide a tractable poxvirus resolvase usable for the development of small molecule inhibitors.

An Assay for Mandelate Racemase Using High-Performance Liquid Chromatography

Mandelate racemase (EC 5.1.2.2) catalyzes the interconversion of the two stereoisomers of mandelic acid. A fixed-time assay for the quantification of mandelate racemase activity has been developed. The assay involves enzymatic conversion ofR-mandelate toS-mandelate (or the reverse reaction) followed by separation and detection of the substrate and product using isocratic reversed-phase high-performance liquid chromatography on a Sumichiral OA-6100 column and absorbance detection. This method offers an economical and efficient alternative to the existing circular dichroism-based and coupled assays.

The effects of ultrasound on the activities of some glycosidase enzymes of industrial importance

The effects of ultrasound on the activities of alpha-amylase and amyloglucosidase toward starch and glycogen hydrolysis, and of invertase toward sucrose hydrolysis, were investigated using an ultrasound bath. A fixed-time assay was employed with product formation determined by reducing equivalents formed per unit time using the 3,5-dinitrosalicylate reagent (DNS). Reaction rates in the presence and absence of ultrasound were compared. Increases in rates were observed in the presence of ultrasound at most concentrations with a marked increase in invertase activity towards sucrose (37% at 0.9 m) and a reduction in the inhibition of sucrose hydrolysis observed at high substrate concentrations. Improvements in the efficiency of mixing and disruption of intra- and intermolecular substrate molecule interactions at high concentrations are possible explanations for the changes observed.

A step forward in enzymatic measurement of creatinine

We describe an improved enzymatic ultraviolet absorbance method for assaying creatinine in serum, plasma, and urine. Creatinine is hydrolyzed by creatinine iminohydrolase (EC 3.5.4.21) to ammonia and N-methylhydantoin. The ammonia produced combines with 2-oxoglutarate and NADPH in the presence of glutamate dehydrogenase to yield glutamate and NADP+. The consumption of NADPH, measured by a two-point fixed-time assay, is proportional to the amount of creatinine in the sample. The assay is carried out in two steps: The first step eliminates background absorbance in hyperlipemic samples and endogenous ammonia through a "clearing system" and an isocitrate dehydrogenase-based "ammonia scavenger system"; the second step starts creatinine measurement. The method affords a simple, rapid, and sensitive assay with good precision and extended linearity; it employs working solutions stable at least 4 months. Test results compare closely with those of the isotope dilution-mass spectrometry Definitive Method, the HPLC procedure, and the fuller's earth method. The proposed method is not subject to interference from several metabolites or from the 72 drugs tested. Because it is easily automated, the method is suitable for routine work in clinical laboratories.

Modulation of PRM Activity by the Lambda PR Promoter in Both the Presence and Absence of Repressor

When the transcription startsites of the phage lambda promoters PRM and PR are separated by 82 bp (the wild-type spacing), mutating PR increases the rate of open complex formation at PRM at all RNA polymerase (RNAP) concentrations tested in vitro. This is reflected in a fourfold increase in kf (the rate constant for isomerization of closed to open complexes) and a threefold decrease in KB (the equilibrium constant for formation of closed complexes). These effects of mutating PR resemble qualitatively those we observed when the separation between the two promoters was decreased by a single base-pair, but are quantitatively less dramatic. Although mutating PR has the additional effect of uncovering a weak promoter, Pα , which overlaps both PRM and PR , the presence of Pα does not account for the effects of PR mutations on open complex formation at PRM.In fixed-time assays at a single RNAP concentration, repressor stimulated PRM approximately threefold on a P-R template, indicating that activation is mediated substantially by a direct interaction between repressor and RNAP. That is, activation of PRM is not merely an indirect consequence of repressing PR. Kinetic data confirm this conclusion. In a P-R genetic backgound, repressor increased kf six- to eightfold and decreased KB approximately twofold. Similar results were obtained when OR3 was mutated, indicating that the effect on KB is not due to repressor binding to OR3. Thus, repressor causes a significant increase in the rate of open complex formation at PRM even when PR is inactive.On a P+R template, 75 nM repressor stimulated PRM by increasing kf eightfold, with no effect on KB, which agrees with previous results. However, increased repressor concentrations stimulated kf by an additional factor of two to four, indicating that previous experiments underestimated the effect of repressor on kf. At the same time, increasing the repressor concentration decreased KB for PRM on a wild-type template. At the highest repressor concentration tested (275 nM), KB decreased 15-fold, presumably due to OR3-mediated repression of PRM . However, at an intermediate repressor concentration (170 nM) values of kf and KB for PRM on a P+R template were in close agreement with the corresponding parameters obtained on a P-R template. These data lead us to suggest that repressor causes a decrease in KB for PRM on both a P+R and a P-R template independent of its ability to bind to OR 3.

Spectrophotometric method to quantify and discriminate urokinase and tissue-type plasminogen activators

Plasminogen activator and urokinase are often used as biological markers of cell activation. However, the methods currently used are cumbersome, make no discrimination between tissue-type plasminogen activator and urokinase, and do not allow expression of the results of the overall reaction in International Units. The one-step method described in this paper lacks these drawbacks. Moreover, we propose use of H- d-Val-Phe-Lys-4-nitroanilide as substrate which has a lower K m than the standard H- d-Val-Leu-Lys-4-nitroanilide which is commercially available. Low concentrations of sodium dodecyl sulfate in the reaction mixture dramatically and preferentially accelerate the reaction catalyzed by tissue-type plasminogen activators. Identical results are obtained under kinetic or fixed-time assay conditions using either a photometer or 96-well plate reader. The corresponding formulae are provided.

A colorimetric assay for the measurement of d-glucose consumption by cultured cells

A colorimetric method is described for measuring glucose consumption by tissue culture cells. This procedure, which utilizes the coupled activities of glucose oxidase and horseradish peroxidase, is insensitive to the spectral interferences caused by the phenol red and sera present in most tissue culture media. The spectral properties (absorbance maxima and apparent absorption coefficients) and stability of a large number of chromogenic horseradish peroxidase substrates were surveyed for their ability to perform in an assay for glucose in the presence of phenol red and sera components. One of these chromophores, the product of an oxidative couple between 4-aminoantipyrine and N-ethyl- N-sulfopropyl- m-toluidine, was subsequently used to develop a fixed time assay for glucose in media samples. The assay required only 10 μl of media in a 1-ml assay volume; reproducibility studies showed variabilities of less than 5% in the assay of a single sample, and values obtained in glucose analyses correlated well with those obtained using commercially available test kits. The assay was used to study the rate of glucose consumption in two different cell types, bovine corneal endothelial cells and human diploid fibroblasts.

Iodometric assay method for beta-lactamase with various beta-lactam antibiotics as substrates.

The rapid fixed-time assay for penicillinase was modified for measuring beta-lactamase activity with twelve substrates, i.e., benzylpenicillin, ampicillin, cloxacillin, methicillin, carbenicillin, cefazolin, cephalothin, cephaloglycin, cephalexin, cephalosporin C, 7-aminocephalosporanic acid, and cefoxitin. The method depends upon the reduction of iodine by the hydrolyzed substrate. Determined experimentally, 1 mol of hydrolyzed penicillins consumed 3.4 to 4.0 mol of iodine (I(2)). Iodine consumption of hydrolyzed cephalosporins varied widely from 1.7 for cephalothin to 3.7 for cefazolin. The method is useful for routine assay of beta-lactamase activity with various substrates.

Assay of tyrosine transaminase activity by conversion of p-hydroxyphenylpyruvate to p-hydroxybenzaldehyde

A rapid, convenient, and highly sensitive fixed-time assay for tyrosine transaminase activity, based on the conversion of p-hydroxyphenylpyruvic acid to p-hydroxybenzaldehyde in strong alkali, is described. Its principal advantage is a 15- to 20-fold increase in sensitivity over the Briggs colorimetric assay commonly employed, and a 2- to 4-fold increase in sensitivity over other methods currently available. Another advantage is its simplicity: the same reagent (NaOH) stops the enzymic reaction and starts the reaction producing the absorbing species, and it is not necessary to centrifuge away a protein precipitate and take an aliquot of a supernatant solution.