A colorimetric method to quantify endo-polygalacturonase activity

We report a new colorimetric assay to quantify endo-polygalacturonase activity, which hydrolyzes polygalacturonic acid to produce smaller chains of galacturonate. Some of the reported polygalacturonase assays measure the activity by detecting the appearance of reducing ends such as the Somogyi-Nelson method. As a result of being general towards reducing groups, the Somogyi-Nelson method is not appropriate when studying polygalacturonase and polygalacturonase inhibitors in plant crude extracts, which often have a strong reducing power. Ruthenium Red is an inorganic dye that binds polygalacturonic acid and causes its precipitation. In the presence of polygalacturonase, polygalacturonic acid is hydrolyzed bringing about a corresponding gain in soluble Ruthenium Red. The described assay utilizes Ruthenium Red as the detection reagent which has been used previously in plate-based assays but not in liquid medium reactions. The new method measures the disappearance of the substrate polygalacturonic acid and is compared to the Somogyi-Nelson assay. The experimental results using lemon peel, a fern fronds and castor leaf crude extracts demonstrate that the new method provides a way to the quickly screening of polygalacturonase activity and polygalacturonase inhibitors in plant crude extracts containing high amounts of reducing power. On the other hand, the Ruthenium Red assay is not able to determine the activity of an exo-polygalacturonase as initial velocity and thus would allow the differentiation between endo- and exo-polygalacturonase activities.     

A novel colorimetric method to quantify tannase activity of viable bacteria

A novel colorimetric method to quantify tannase activity of viable tannase-producing bacterial strains was developed through application of a visual reading method that was to detect the activity qualitatively. The novel method was sensitive enough to quantify the marginal tannase activity of strains that could not be otherwise measured by conventional spectrophotometric or colorimetric methods.     

A multiplexed homogeneous fluorescence-based assay for protein kinase activity in cell lysates

New methods to quantify protein kinase activities directly from complex cellular mixtures are critical for understanding biological regulatory pathways. Herein, a fluorescence-based chemosensor strategy for the direct measurement of kinase activities in crude mammalian cell lysates is described. We first designed a new fluorescent peptide reporter substrate for each target kinase. These kinase chemosensors were readily phosphorylated by recombinant target enzyme and underwent a several-fold fluorescence increase upon phosphorylation. Then, using unfractionated cell lysates, a homogeneous kinase assay was developed that was reproducible, linear and highly preferential for monitoring changes in cellular activity of the target kinase. The general protocol was developed for the kinase Akt and then easily extended to measure protein kinase A (PKA) and mitogen-activated protein kinase-associated protein kinase 2 (MK2) activities. This assay platform is immediately useful for studying protein kinase signaling in crude cellular extracts.     

Sandwich hybridisation assay for quantitative detection of yeast RNAs in crude cell lysates

Background  

A rapid microtiter plate based sandwich hybridization assay was developed for detection and quantification of single RNA species using magnetic beads. Following solution hybridization target RNA molecules were collected by biotin-streptavidin affinity binding and detected by fluorescence signal generated by alkaline phosphatase. The 18S rRNA and SUC2 mRNA of Saccharomyces cerevisiae were used as model RNA target molecules.     

A sensitive colorimetric assay for mitochondrial alpha-glycerophosphate dehydrogenase

A simple reproducible assay for mitochondrial α-glycerophosphate dehydrogenase (GPD) has been described. It takes advantage of the ability of 2-p-iodo-3-p-nitro-5-phenyl tetrazolium chloride (INT) to directly accept electrons from the dehydrogenase. The assay will accurately measure the enzyme using 20 μg of mitochondrial protein, but it could be scaled down further using smaller volumes and microcuvettes.     

Kinetic assay for characterization of spleen tyrosine kinase activity and inhibition with recombinant kinase and crude cell lysates

Spleen tyrosine kinase (Syk) is involved in the activation of cells implicated in allergic or autoimmune diseases and certain cancers. Therefore, Syk inhibitors may prove to be effective in treating diseases where Syk activity or expression is increased or deregulated. We developed a continuous and direct (noncoupled) fluorescence intensity assay for measuring Syk activity using purified recombinant enzyme or crude lysates generated from anti-immunoglobulin M (IgM) antibody-treated RAMOS cells. The assay is based on the chelation-enhanced fluorophore 8-hydroxy-5-(N,N-dimethylsulfonamido)-2-methylquinoline (referred to as Sox), which has been incorporated into a peptide substrate selected for robust detection of Syk activity. This homogeneous assay is simple to use, provides considerably more information, and has been adapted to a 384-well, low-volume microtiter plate format that can be used for the high-throughput identification and kinetic characterization of Syk inhibitors. The assay can be performed with a wide range of adenosine triphosphate (ATP) concentrations and, therefore, can be used to analyze ATP-competitive and ATP-noncompetitive/allosteric kinase inhibitors. Measurement of Syk activity in RAMOS crude cell lysates or immunoprecipitation (IP) capture formats may serve as a physiologically more relevant enzyme source. These Sox-based continuous and homogeneous assays provide a valuable set of tools for studying Syk signaling and for defining inhibitors that may be more effective in controlling disease.     

A colorimetric assay for screening transketolase activity

A tetrazolium red-based colorimetric assay has been devised to screen for transketolase activity with a range of aldehyde acceptors. The colorimetric TK assay is able to detect >8% bioconversion using non-alpha-hydroxylated aldehydes as acceptor substrates and is significantly faster and more convenient to use than chromatographic procedures.     

A direct and continuous assay for the determination of thioredoxin reductase activity in cell lysates

Thioredoxin reductase (TR) is an oxidoreductase responsible for maintaining thioredoxin in the reduced state, thereby contributing to proper cellular redox homeostasis. The C-terminal active site of mammalian TR contains the rare amino acid selenocysteine, which is essential to its activity. Alterations in TR activity due to changes in cellular redox homeostasis are found in clinical conditions such as cancer, viral infection, and various inflammatory processes; therefore, quantification of thioredoxin activity can be a valuable indicator of clinical conditions. Here we describe a new direct assay, termed the SC–TR assay, to determine the activity of TR based on the reduction of selenocystine, a diselenide-bridged amino acid. Rather than being an end-point assay as in older methods, the SC–TR assay directly monitors the continuous consumption of NADPH at 340 nm by TR as it reduces selenocystine. The SC–TR assay can be used in a cuvette using traditional spectrophotometry or as a 96-well plate-based format using a plate reader. In addition, the SC–TR assay is compatible with the use of nonionic detergents, making it more versatile than other methods using cell lysates.     

Cycling of NADPH by glucose 6-phosphate dehydrogenase optimizes the spectrophotometric assay of nitric oxide synthase activity in cell lysates.

The measurement of nitric oxide synthase activity in cell lysates is often performed by radiochemical assay that quantifies the conversion of L-[3H]arginine to L-[3H]citrulline. We have developed a spectrophotometric procedure which continuously recycles NADPH through the addition of glucose 6-phosphate dehydrogenase to the cell lysate. This allows nitric oxide synthase to operate linearly for hours, so that nitric oxide-derived nitrite accumulates at amounts sufficient to be detected with the Griess assay. The incorporation of cycling of NADPH also improves the radiochemical assay for nitric oxide synthase activity.     

A rapid colorimetric assay for heparinase activity.

A rapid, sensitive, assay for enzymes that degrade heparin is described. The procedure is based on the interference of heparin with color development during the interaction of protein with the dye Coomassie brilliant blue. The loss of this property when the glycosaminoglycan is degraded by heparinase can be used to quantify activity of the enzyme in pure form, or in complex biological samples such as tissue homogenates or serum. The assay is also suitable for studying dependence of heparinase activity under conditions such as varying pH and temperature.     

Dot assay for neomycin phosphotransferase activity in crude cell extracts

A dot assay for determining neomycin phosphotransferase (NPT II) activity in crude cell extracts has been developed. The assay provides for the rapid screening of large numbers of cell cultures generated in gene transformation experiments using NPT II as a dominant selectable marker. Currently, the commonly used procedure for NPT II assay employs a time-consuming electrophoretic protein separation step to eliminate a positive interference resulting from putative protein kinase activities present in crude cell extracts. The dot method we have developed is based upon the ability of nitrocellulose membrane to eliminate that positive interference without a prior protein separation step. It provides a sensitive, reproducible, and significantly more convenient and rapid means of screening large numbers of cell extracts in order to distinguish cultures producing high levels of NPT II from those that do not.     

The synthetic peptide RPRAATF allows specific assay of Akt activity in cell lysates

The Akt protein kinase is a critical signaling molecule in a range of cellular processes. A key to identifying the role of this pleiotropic kinase in any particular process is the ability to quantitate its activity. In this study we show that the synthetic peptide RPRAATF is a specific substrate for the kinase in crude cell extracts, thus enabling rapid, convenient, and sensitive assay of Akt activity. Peptide kinase activity was confined to a single peak upon sequential ion-exchange chromatography of whole-cell extracts of Balb/c 3T3 fibroblasts. This activity was stimulated by both platelet-derived growth factor and pervanadate, phosphatidyl inositol 3-kinase dependent, and inhibited by specific immunodepletion with anti-Akt antisera. Furthermore, direct assays of crude extracts from a range of cell types using this peptide were consistent with the results obtained using specific immunoprecipitation assays.     

A high-throughput colorimetric assay to measure the activity of glutamate decarboxylase

A pH-sensitive colorimetric assay has been established to quantitatively measure glutamate decarboxylase (GAD) activity in bacterial cell extracts using a microplate format. GAD catalyzes the irreversible α-decarboxylation of l-glutamate to γ-aminobutyrate. The assay is based on the color change of bromocresol green due to an increase in pH as protons are consumed during the enzyme-catalyzed reaction. Bromocresol green was chosen as the indicator because it has a similar pK_a to the acetate buffer used. The corresponding absorbance change at 620 nm was recorded with a microplate reader as the reaction proceeded. A difference in the enzyme preparation pH and optimal pH for GAD activity of 2.5 did not prevent this method from successfully allowing the determination of reaction kinetic parameters and the detection of improvements in enzymatic activity with a low coefficient of variance. Our assay is simple, rapid, requires minimal sample concentration and can be carried out in robotic high-throughput devices used as standard in directed evolution experiments. In addition, it is also applicable to other reactions that involve a change in pH.     

High-precision fluorescence assay for sphingomyelinase activity of isolated enzymes and cell lysates

Sphingomyelinases are important enzymes of signal transduction. They catalyze the hydrolysis of sphingomyelin, giving rise to the intracellular formation of biologically active ceramide and phosphatidylcholine. Here we report on a fluorescence method for the fast and accurate determination of this enzyme in biological samples. The assay is based on a fluorescent sphingomyelin analog carrying fluorescent 7-nitro-2-1,3-benzooxadiazolyl amino-dodecanoic acid instead of an aliphatic acyl chain at the nitrogen atom. The fluorescent substrate is hydrolysed by sphingomyelinases to form fluorescent ceramide, which can be separated from the remaining substrate using TLC on silica gel. The fluorescence intensity pattern obtained on the TLC plate can accurately be determined using a CCD camera. Typically, a large number of samples can be analyzed simultaneously. Examples for the quantitative analysis of sphingomyelinases from freshly prepared cellular homogenates as well as from commercial sources are given.     

Quantitative in situ gel electrophoretic assay for neomycin phosphotransferase activity in mammalian cell lysates

A method for the assay of neomycin phosphotransferase activity in eucaryotic cell lysates is described. Total cytoplasmic proteins are fractionated in nondenaturing polyacrylamide gels and then allowed to react in situ with [gamma-32P]ATP and kanamycin. The reaction products are detected by blotting to phosphocellulose paper and autoradiography. The assay is linear with protein concentration and sensitive enough to detect expression in transient assays.     

A rapid colorimetric assay of killer toxin activity in yeast

Abstract The pale yellow redox indicator 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) is reduced to a dark blue end-product, MTT-Formazan, by the mitochondrial dehydrogenases of living cells. MTT reduction can be measured spectrophotometrically at a wavelength of 570 nm and a method is described to assay the cidal activity of Williopsis mrakii killer toxin against sensitive cells of Candida glabrata . The MTT assay is rapid, quantitative and compares favourably with traditional plating techniques for the assessment of sensitive viability.     

PCR amplification of streptococcal DNA using crude cell lysates

Gram-positive organisms such as streptococci and enterococci are often difficult to lyse. Obtaining DNA for procedures such as PCR amplification usually requires a large scale isolation for each strain under investigation. We describe a simple procedure for small volumes of whole cells, involving pretreatment with detergent and proteinase that allows for efficient release of DNA for PCR amplification. This procedure is fast, reproducible, can be used with a large number of samples, and has been successfully applied to a variety of streptococcal and enterococcal strains.     

A colorimetric assay for sulfiredoxin activity using inorganic phosphate measurement

2-Cys peroxiredoxin (Prx) is the major subgroup of a family of Prx enzymes that reduce peroxide molecules such as hydrogen peroxide (H₂O₂). 2-Cys Prxs are inactivated when their active site cysteine residue is hyperoxidized to sulfinic acid. Sulfiredoxin (Srx) is an enzyme that catalyzes reduction of hyperoxidized 2-Cys Prxs in the presence of ATP, Mg²⁺, and thiol equivalent. Therefore, Srx activity is crucial for cellular function of 2-Cys Prxs. The method currently available for the determination of Srx activity relies on immunoblot detection using antibodies to hyperoxidized enzymes. Here we introduce a simple quantitative assay for Srx activity based on the colorimetric determination of inorganic phosphate released in Srx-dependent reduction of hyperoxidized Prx using the malachite green. The colorimetric assay was used for high-throughput screening of 25,000 chemicals to find Srx inhibitors.     

A malachite green colorimetric assay for protein phosphatase activity.

A simple and sensitive colorimetric assay for protein phosphatase activity based on the determination of released Pi by an improved malachite green procedure (A. A. Baykov, O. A. Evtushenko, and S.M. Avaeva, 1988, Anal. Biochem. 171, 266-270) is described. Proteins must be removed or stabilized prior to Pi determination with 0.25 N sulfuric acid or 3% (w/w) perchloric acid. Alternatively, to avoid possible acid hydrolysis of phosphate groups from organic compounds during deproteinization, the protein present in the phosphatase assay mixture can be stabilized with sodium dodecyl sulfate. In this case, the excess detergent is subsequently removed by precipitation with KCl because it colors with the malachite green reagent. The above procedure was applied to the determination of phosphorylase phosphatase activity in bovine brain extracts and the results are comparable to those obtained with the radioisotopic phosphatase assay.