A continuous spectrophotometric enzyme-coupled assay for deoxynucleoside triphosphate triphosphohydrolases

We describe a continuous, spectrophotometric, enzyme-coupled assay useful to monitor reactions catalyzed by nucleoside triphosphohydrolases. In particular, using Escherichia coli deoxynucleoside triphosphohydrolase (Dgt), which hydrolyzes dGTP to deoxyguanosine and tripolyphosphate (PPP_i) as the enzyme to be tested, we devised a procedure relying on purine nucleoside phosphorylase (PNPase) and xanthine oxidase (XOD) as the auxiliary enzymes. The deoxyguanosine released by Dgt can indeed be conveniently subjected to phosphorolysis by PNPase, yielding deoxyribose-1-phosphate and guanine, which in turn can be oxidized to 8-oxoguanine by XOD. By this means, it was possible to continuously detect Dgt activity at 297 nm, at which wavelength the difference between the molar extinction coefficients of 8-oxoguanine (8000 M⁻¹ cm⁻¹) and guanine (1090 M⁻¹ cm⁻¹) is maximal. The initial velocities of Dgt-catalyzed reactions were then determined in parallel with the enzyme-coupled assay and with a discontinuous high-performance liquid chromatography (HPLC) method able to selectively detect deoxyguanosine. Under appropriate conditions of excess auxiliary enzymes, the activities determined with our continuous enzyme-coupled assay were quantitatively comparable to those observed with the HPLC method. Moreover, the enzyme-coupled assay proved to be more sensitive than the chromatographic procedure, permitting reliable detection of Dgt activity at low dGTP substrate concentrations.     

Sensitive PCR Detection of Meloidogyne arenaria, M. incognita, and M. javanica Extracted from Soil

We have developed a simple PCR assay protocol for detection of the root-knot nematode (RKN) species Meloidogyne arenaria, M. incognita, and M. javanica extracted from soil. Nematodes are extracted from soil using Baermann funnels and centrifugal flotation. The nematode-containing fraction is then digested with proteinase K, and a PCR assay is carried out with primers specific for this group of RKN and with universal primers spanning the ITS of rRNA genes. The presence of RKN J2 can be detected among large numbers of other plant-parasitic and free-living nematodes. The procedure was tested with several soil types and crops from different locations and was found to be sensitive and accurate. Analysis of unknowns and spiked soil samples indicated that detection sensitivity was the same as or higher than by microscopic examination.     

Radioimmunoassay for quantitative determination of morphine in capsules of Papaver somniferum

A radioimmunoassay (RIA) procedure for the determination of pmol quantitites of morphine in capsule samples of Papaver somniferum was developed. An antiserum developed against a conjugate of morphine-3-hemisuccinate-BSA was relatively specific for morphine and possessed moderated cross-reactivity with codeine and mild cross-reactivity with thebaine, but none with narceine, papaverine, or noscapine. The standard curve was linear over a range of 0.01–0.20 ng. This assay allows for the rapid, sensitive and precise determination of morphine in unpurified aqueous extracts of capsule samples. The amounts of morphine in the aqueous extracts determined by radioimmunoassay were validated by high performance liquid chromatography (HPLC). The two methods show a high correlation coefficient (r = 0.98) with no significant difference in determinations of morphine content by RIA and HPLC.     

Cytokinin profiling in plant tissues using ultra-performance liquid chromatography-electrospray tandem mass spectrometry

We have developed a simple, high-throughput batch immunoextraction (IAE) micropurification procedure for extracting a wide range of naturally occurring cytokinins (bases, ribosides, O- and N-glucosides, and nucleotides) from plant tissues in solutions that are compatible with ultra-performance liquid chromatography (UPLC), thereby facilitating sensitive subsequent analysis. The UPLC system was coupled to a tandem quadrupole mass spectrometer (MS/MS) equipped with an electrospray interface (ESI). Small (mg) amounts of tissues were purified by solid-phase extraction (SPE) followed by an immunoaffinity clean-up step and two fast chromatographic separations of most cytokinin metabolites (bases, ribosides, and 9-glucosides in the first, O-glucosides and nucleotides in the second). Using UPLC, the runs were up to 4-fold faster than in standard cytokinin analyses, and both retention times and injection volumes were less variable (RSDs, 0.15-0.3% and 1.0-5.5%, respectively). In multiple reaction monitoring (MRM) mode, the detection limit for most of the cytokinins analyzed was close to 1 fmol (5-25 fmol for O-glucosides and nucleotides) and the linear range spanned at least five orders of magnitude. The extraction and purification method was optimized using poplar (Populus × canadensis Moench, cv Robusta) leaf samples, and the analytical accuracy was further validated using IAE-purified 10-day-old Arabidopsis thaliana plants spiked with 1 and 10 pmol of cytokinin derivatives. This approach can be used for rapid, sensitive qualitative and/or quantitative analysis of more than 50 natural cytokinins in minute amounts of plant tissues with high performance, robustness, and accuracy.     

Carboxylic Acids as Biomarkers of Biomphalaria alexandrina Snails Infected with Schistosoma mansoni

Biomphalaria alexandrina snails play an indispensable role in transmission of schistosomiasis. Infection rates in field populations of snails are routinely determined by cercarial shedding neglecting prepatent snail infections, because of lack of a suitable method for diagnosis. The present study aimed at separation and quantification of oxalic, malic, acetic, pyruvic, and fumaric acids using ion-suppression reversed-phase high performance liquid chromatography (HPLC) to test the potentiality of these acids to be used as diagnostic and therapeutic biomarkers. The assay was done in both hemolymph and digestive gland-gonad complex (DGG) samples in a total of 300 B. alexandrina snails. All of the studied acids in both the hemolymph and tissue samples except for the fumaric acid in hemolymph appeared to be good diagnostic biomarkers as they provide not only a good discrimination between the infected snails from the control but also between the studied stages of infection from each other. The most sensitive discriminating acid was malic acid in hemolymph samples as it showed the highest F-ratio. Using the Z-score, malic acid was found to be a good potential therapeutic biomarker in the prepatency stage, oxalic acid and acetic acid in the stage of patency, and malic acid and acetic acid at 2 weeks after patency. Quantification of carboxylic acids, using HPLC strategy, was fast, easy, and accurate in prediction of infected and uninfected snails and possibly to detect the stage of infection. It seems also useful for detection of the most suitable acids to be used as drug targets.     

A simple method for the extraction and quantification of photopigments from Symbiodinium spp.

We have developed a simple, mild extraction procedure using methanol which, when coupled with HPLC analysis and diode array detection (DAD), can be used to quantify the major photopigments found in cultured Symbiodinium spp. Extracts were prepared by suspending, fresh or frozen (− 70 °C), wet cell pellets in methanol and sonicating or not sonicating the cell suspensions before soaking the cells for 2 h in an ice bath. To assist the soaking process, cell suspensions were vortex mixed at 30 min intervals. After soaking, 0.5 M ammonium acetate buffer was added (1 part buffer to 9 parts methanol) before suspensions were stored over night at − 20 °C. Greater than 92% the recoverable pigment was obtained in the initial extraction of the four major photopigments, chlorophyll c, peridinin, diadinoxanthin, and chlorophyll a. Neither sonication nor freezing substantially increased the recovery of photopigments extracted with methanol. Extraction by other commonly used solvents such as acetone or acetone:water with or without freezing and sonication were less effective.     

Analysis of polyamines in higher plants by high performance liquid chromatography

A sensitive (0.01-1 nmol) method has been developed for the analysis of polyamines in higher plant extracts based on high performance liquid chromatography (HPLC) of their benzoyl derivatives (Redmond, Tseng 1979 J Chromatogr 170: 479-481). Putrescine, cadaverine, agmatine, spermidine, spermine, and the less common polyamines nor-spermidine and homospermidine can be completely resolved by reverse phase HPLC, isocratic elution with methanol:water (64%, v/v) through a 5-μm C₁₈ column, and detection at 254 nm. The method can be directly applied to crude plant extracts, and it is not subject to interference by carbohydrates and phenolics. A good quantitative correlation was found between HPLC analysis of benzoylpolyamines and thin layer chromatography of their dansyl derivatives. With the HPLC method, polyamine titers have been reproducibly estimated for various organs of amaranth, Lemna, oat, pea, Pharbitis, and potato. The analyses correlate well with results of thin layer chromatography determinations.     

Comparison of high-performance liquid chromatography and fluorophore-assisted carbohydrate electrophoresis methods for analyzing peptidoglycan composition of Escherichia coli

Currently, reversed-phase high-performance liquid chromatography (HPLC) is the method of choice for determining the types and amounts of muropeptide subunits comprising bacterial peptidoglycan. Although effective and sensitive, the technique does not lend itself to high throughput screening, and its complexity and equipment requirements may dissuade some investigators from pursuing certain types of cell wall experiments. Previously, we showed that muropeptides can be labeled with a fluorescent dye and separated by fluorophore-assisted carbohydrate electrophoresis (FACE), a simple and rapid gel procedure that might serve as a prelude to more intense analysis by HPLC. To validate the utility of FACE, we used both techniques to perform a side-by-side analysis of the peptidoglycan of eight mutants and their Escherichia coli parent strain. FACE and HPLC both detected the seven major muropeptides, which represent more than 95% of the total muropeptides present in this organism. In addition, FACE returned the same relative and quantitative results in 92% of 72 measurements, indicating that the procedure gives an accurate overview of peptidoglycan composition. The results also suggest a possible biochemical activity for the AmpC and AmpH proteins of E. coli, and the use of FACE as an in vitro enzyme assay detected possible substrate preferences for the endopeptidase penicillin binding protein 4.     

Qualitative and quantitative determination of major saponins in Paris and Trillium by HPLC-ELSD and HPLC–MS/MS

High-performance liquid chromatographic (HPLC) with evaporative light scattering detection (ELSD) and HPLC with electrospray ionization multistage tandem mass spectrometry (HPLC–ESI-MSⁿ) were used to identify and quantify steroid saponins in Paris and Trillium plants. The content of the known saponins such as Paris I, II, III, V, VI, VII, H, gracillin and protodioscin in Paris and Trillium plants was determined simultaneously using the developed HPLC-ELSD method. Furthermore, other 12 steroid saponins were identified by HPLC–ESI(+/−)-MSⁿ detection. In the end, a developed analytical procedure was proved to be a reliable and rapid method for the quality control of Paris and Trillium plants. In addition, the alternative resources for Paris yunnanensis used as a traditional Chinese medicine were discovered according to the hierarchical clustering analysis of the saponin fraction of these plants.     

Nonradioactive enzyme measurement by high-performance liquid chromatography of partially purified sugar-1-kinase (glucuronokinase) from pollen of Lilium longiflorum

Here we present a highly sensitive and simple high-performance liquid chromatography (HPLC) method that enables specific quantification of glucuronokinase activity in partially purified extracts from pollen of Lilium longiflorum without radioactive labeled substrates. This assay uses a recombinant UDP-sugar pyrophosphorylase with broad substrate specificity from Pisum sativum (PsUSP) or Arabidopsis thaliana (AtUSP) as a coupling enzyme. Glucuronokinase was partially purified on a DEAE-sepharose column. Kinase activity was measured by a nonradioactive coupled enzyme assay in which glucuronic acid-1-phosphate, produced in this reaction, is used by UDP-sugar pyrophosphorylase and further converted to UDP-glucuronic acid. This UDP-sugar, as well as different by-products, is detected by HPLC with either a strong anion exchange column or a reversed phase C18 column at a wavelength of 260 nm. This assay is adaptive to different kinases and sugars because of the broad substrate specificity of USP. The HPLC method is highly sensitive and allows measurement of kinase activity in the range of pmol min^-1. Furthermore, it can be used for determination of pure kinases as well as crude or partially purified enzyme solutions without any interfering background from ATPases or NADH oxidizing enzymes, known to cause trouble in different photometric assays.     

A simplified and sensitive fluorescent method for disaccharide analysis of both heparan sulfate and chondroitin/dermatan sulfates from biological samples

Sulfated glycosaminoglycans regulate the biological functions of a wide variety of proteins, primarily through high affinity interactions mediated by specific sugar sequences or patterns/densities of sulfation. Disaccharide analysis of such glycosaminoglycans yields important diagnostic and comparative structural information on sulfate patterning. When applied to specific oligosaccharides it can also make a vital contribution to sequence elucidation. Standard UV detection of lyase-generated disaccharides resolved by HPLC can lack sufficient sensitivity and be compromised by contaminating UV signals, when dealing with scarce tissue- or cell culture-derived material. Various methods exist for improved detection, but usually involve additional HPLC hardware and often necessitate different procedures for analyzing different glycosaminoglycans. We describe a simple procedure, requiring only standard HPLC instrumentation, involving prederivatization of disaccharides with 2-aminoacridone with no cleanup of samples, followed by a separation by reverse-phase HPLC that is sensitive to as little as approximately 100 pg (approximately 10(-13) mol) of an individual disaccharide, thereby allowing analyses of >10 ng of total glycosaminoglycan. Importantly, separate analysis of both HS/heparin and CS/DS species within a mixed glycosaminoglycan pool can be performed using the same procedure on a single column. We demonstrate its applicability in dealing with small quantities of material derived from rat liver (where we demonstrate a high abundance of the unusual CS-E species within the CS/DS pool) and MDCK cells (which revealed a HS species of relatively low N-sulfation, but high O-sulfation). This simplified method should find a widespread utility for analyzing glycosaminoglycans from limited animal and cell culture samples.     

Solid-phase sequencing on the gas-phase sequencer

Automated Edman degradation has been successfully used for determining the primary structure of numerous peptides and proteins. Quantitative solid-phase Edman degradation has great potential use for amino acid sequence analysis of synthetic peptides assembled on resin support by the Merrifield procedure. We report here the combined use of a modified gas-phase sequencer program and our improved reversed-phase HPLC analysis for PTH-amino acids to carry out the sequence analysis on synthesized peptide resins. This approach is far more sensitive than using glass beads on the conventional solid-phase sequencer. The peptide was assembled on copoly (styrene-1% divinylbenzene) resin beads at an initial substitution of 0.54 mmol/g. On a routine basis, 10-15 resin beads are used, and a repetitive yield of 94% is obtained: as few as 4 beads can be successfully sequenced. The HPLC PTH-amino acid analysis is sensitive down to subpicomole quantities. This procedure offers a sensitive and rapid analytical tool for checking the purity of peptides as they are being assembled on solid support.     

3,4-Dihydroxy-l-phenylalanine as a biomarker of oxidative damage in proteins: Improved detection using cloud-point extraction and HPLC

Oxidized protein adducts are formed under conditions of oxidative stress and may represent a valuable biomarker for a variety of diseases which share this common aetiology. A suitable candidate biomarker for oxidized proteins is protein-bound 3,4-dihydroxyl-l-phenylalanine (l-DOPA), which is formed on 3′-hydroxylation of tyrosine residues by hydroxyl radicals. Existing methodologies to measure protein-bound l-DOPA employ lengthy acid hydrolysis steps (ca. 16 h) which may cause artifactual protein oxidation, followed by HPLC with detection based on the intrinsic fluorescence of l-DOPA. We report a novel method for the measurement of protein-bound l-DOPA which involves rapid hydrolysis followed by pre-column concentration of 6-aminoquinolyl-derivatives using cloud-point extraction. The derivatized material is resolved by reversed-phase HPLC in less than 30 min and has derivatization chemistry compatible with both UV and fluorescent detection, providing detection down to the femtomole level. The method provides identical results to those found with highly specific ELISA-based techniques and requires only basic instrumentation. The stability of the 6-aminoquinolyl-derivatives together with the fast and sensitive nature of the assay will be appealing to those who require large sample throughput.     

Improved Method for HPLC Analysis of Polyamines, Agmatine and Aromatic Monoamines in Plant Tissue

The high performance liquid chromatographic (HPLC) method of Flores and Galston (1982 Plant Physiol 69: 701) for the separation and quantitation of benzoylated polyamines in plant tissues has been widely adopted by other workers. However, due to previously unrecognized problems associated with the derivatization of agmatine, this important intermediate in plant polyamine metabolism cannot be quantitated using this method. Also, two polyamines, putrescine and diaminopropane, also are not well resolved using this method. A simple modification of the original HPLC procedure greatly improves the separation and quantitation of these amines, and further allows the simulation analysis of phenethylamine and tyramine, which are major monoamine constituents of tobacco and other plant tissues. We have used this modified HPLC method to characterize amine titers in suspension cultured carrot (Daucus carota L.) cells and tobacco (Nicotiana tabacum L.) leaf tissues.     

Highly sensitive assay for tyrosine hydroxylase activity by high-performance liquid chromatography

A highly sensitive assay for tyrosine hydroxylase (TH) activity by high-performance liquid chromatography (HPLC) with amperometric detection was devised based on the rapid isolation of enzymatically formed DOPA by a double-column procedure, the columns fitted together sequentially (the top column of Amberlite CG-50 and the bottom column of aluminium oxide). DOPA was adsorbed on the second aluminium oxide column, then eluted with 0.5 M hydrochloric acid, and assayed by HPLC with amperometric detection. d-Tyrosine was used for the control. α-Methyldopa was added to the incubation mixture as an internal standard after incubation. This assay was more sensitive than radioassays and 5 pmol of DOPA formed enzymatically could be measured in the presence of saturating concentrations of tyrosine and 6-methyltetrahydropterin. The TH activity in 2 mg of human putamen could be easily measured, and this method was found to be particularly suitable for the assay of TH activity in a small number of nuclei from animal and human brain.     

Quantification of Invasion of Two Strains of Steinernema carpocapsae (Weiser) into Three Lepidopteran Larvae

Studies with last instar larvae of the fall armyworm, Spodoptera frugiperda (J. E. Smith), the black cutworm, Agrotis ipsilon (Hufnagel), and the greater wax moth, Galleria mellonella (L.) were used to quantify the invasive ability of two strains (All and Mexican) of Steinernema carpocapsae and to determine how factors in the bioassay procedure affect both nematode invasion and host mortality. Nematode invasive ability was variable, with 10-50% of nematodes successfully infecting the host. The percentage of infectives invading the host (invasion efficiency) was positively related to increases in length of host exposure time and number of hosts per arena, negatively related to increases in substrate surface area per host, and not affected by nematode concentration. There was a direct relationship between concentration applied and the number of nematodes invading the host. Mortality was less affected than invasion efficiency by bioassay conditions and appears to be a much less sensitive index of nematode activity than invasive ability.     

A new modified Edman procedure for analysis of N-terminal valine adducts in hemoglobin by LC–MS/MS

A rapid and sensitive method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) for simultaneous determination of adducts from acrylamide, glycidamide and ethylene oxide to N-terminal valines in hemoglobin (Hb) was developed. This new procedure is based on the same principles as the N-alkyl Edman procedure for analysis of adducts from electrophilic agents to N-terminal valines in Hb. The N-substituted valines can be detached, enriched and measured selectively as thiohydantoins by the use of an Edman reagent, in this case fluorescein isothiocyanate (FITC). This procedure is denoted as the “adduct FIRE procedure” as the FITC reagent is used for measurement of adducts (R) formed from electrophilic compounds with a modified Edman procedure. In this study, fluorescein thiohydantoin (FTH) analytes of N-substituted valines from acrylamide, glycidamide and ethylene oxide, as well as their corresponding hepta- and tri-deuterium-substituted analogues, were synthesized. These analytes (n = 8) were then characterized by LC–MS/MS (ESI, positive ion mode) and obtained product ions were interpreted. A considerable work with optimization of the FIRE procedure™, resulted in a procedure in which low background levels of the studied adducts could be measured from 250 μL lyzed whole blood samples (human non-smokers). The analytes were enriched and purified with solid phase extraction columns and analyzed by LC–MS/MS with LOQ down to 1 pmol adduct/g Hb. Compared to other procedures for determination of N-terminal Hb adducts, the introduction of FITC has led to a simplified procedure, where whole blood also can be used, giving new opportunities and reduced hand on time with increased sample throughput.     

Leishmania (V.) braziliensis: detection by PCR in biopsies from patients with cutaneous leishmaniasis

Cutaneous leishmaniases present similar clinical appearances, but differing prognosis in the course of infection. Ulcers caused by parasites of the subgenus Viannia are more aggressive than ulcers caused by parasites of the subgenus Leishmania. Another problem is distinguishing between true Leishmania infection and other skin diseases in endemic areas, where cutaneous lesions and a single positive Montenegro intradermal test are enough to submit patients to specific treatment for cutaneous leishmaniasis. This study evaluated the efficacy of PCR in detecting in Leishmania in patients with cutaneous lesions. Leishmania (V.) braziliensis complex was determined by a primer pair from the multicopy spliced leader RNA. The results were compared to those of traditional methods. We analyzed biopsies of 109 patients with cutaneous lesions in the second most endemic region of Sao Paulo State, Brazil. Definitive diagnosis was established by clinical and “consensus laboratory criteria” (positive culture, stained tissue smears or PCR). Of 52 patients with cutaneous leishmaniasis, 96% had positive PCR, 69%, positive parasitological tests and 100%, positive Montenegro intradermal tests. Histopathological examination (only in 32 samples) were positive in 14 samples, suggestive in 14 and negative in 4 samples. All 57 patients with other etiologies had negative results in parasitological methods, PCR and histopathological examination (in 39 samples), but Montenegro intradermal tests were positive in 35%. PCR was highly sensitive and specific for L. (V.) braziliensis complex detection compared with other laboratory methods. Despite the specificity of the parasitological tests, the sensitivity was less than 70%. Montenegro intradermal reaction was highly sensitive, but with low specificity, only 65%. As suggestive results in histopathological examinations were shown in 14 samples, it was difficult to determine the true result. PCR applied to biopsies proved to be useful for differential diagnosis of cutaneous lesions of other etiologies in patients living in endemic areas. The advantages are most striking in clinical specimens with scarce amastigotes for which conventional methods have low sensitivity and should be considered for clinical and epidemiological patterns. On the other hand, both Montenegro intradermal test and parasitological methods are only modestly effective in cutaneous leishmaniasis diagnosis.     

Immunoaffinity co-purification of cytokinins and analysis by high-performance liquid chromatography with ultraviolet-spectrum detection

A rapid methodology for the simultaneous analysis of a large number of cytokinins is presented. The cross-reactivity of a mixture of polyclonal antibodies against zeatin riboside and isopentenyladenosine was exploited in a protocol that can be used for immunoaffinity purification of 23 additional cytokinins. Ligands include the cytokinin bases zeatin, dihydrozeatin, isopentenyladenine, benzyl-adenine and kinetin, and their corresponding nucleoside, nucleoside-5′-monophosphate, and 9-glucoside derivatives, as well as cis-zeatin, cis-zeatin riboside, the 2-methylthiol derivatives of isopentenyladenosine and zeatin riboside, and benzyl-adenine-3-glucoside. Mixtures of cytokinins could be retained with high recoveries of all the components. Immunoaffinity purification of extracts of Arabidopsis thaliana (L.) Heynh. and Solarium tuberosum L. gave fractions clean enough, as verified by gas chromatographymass spectrometry (GC-MS), to allow analysis of endogenous cytokinins using a single high-performance liquid chromatography (HPLC) step with on-line UV-spectrum detection. The detection limit was 4–6 pmol. The procedure described forms a routine assaying technique that is faster and simpler, yet yields better qualitative and quantitative information than the commonly used procedure of immunoassaying of HPLC fractions.     

The improvement of a phenotype microarray protocol for the chemical sensitivity analysis of Streptococcus thermophilus

Phenotype MicroArray (PM) permits the characterisation of bacteria under nearly 2000 culture conditions. The PM standard procedure for the chemical sensitivity analysis of Gram-positive bacteria failed in the analysis of Streptococcus thermophilus. Therefore, we developed an efficient and reproducible protocol to obtain a chemically sensitive profile of S. thermophilus using PM.