المؤلفون: Jain, Priyamvada¹, Das, Smita¹, Chakma, Babina¹, Goswami, Pranab¹ pgoswami@iitg.ernet.in

المصدر: Analytical Biochemistry. Dec2016, Vol. 514, p32-37. 6p.

مصطلحات موضوعية: *APTAMERS, *GRAPHENE oxide, *LACTATE dehydrogenase, *RAMAN spectra, *STATISTICAL reliability

مستخلص: A 90 mer ssDNA aptamer (P38) enriched against Plasmodium falciparum lactate dehydrogenase (PfLDH) through SELEX process was immobilized over glassy carbon electrode (GCE) using graphene oxide (GO) as an immobilization matrix, and the modified electrode was investigated for detection of PfLDH. The GO was synthesized from powdered pencil graphite and characterized by XRD based on the increased interlayer distance between graphitic layers from 0.345 nm for graphite to 0.829 nm for GO. The immobilization of P38 on GO was confirmed by I D /I G intensity ratio in Raman spectra where, the ratio were 0.67, 0.915, and 1.35 for graphite, GO and P38-GO, respectively. The formation of the P38 layer over GO-GCE was evident from an increase in the surface height in AFM analysis of the electrode from ∼3.5 nm for GO-GCE to ∼27 nm for P38-GO-GCE. The developed aptasensor when challenged with the target, a detection of as low as 0.5 fM of PfLDH was demonstrated. The specificity of the aptasensor was confirmed through a voltametric measurement at 0.65 V of the reduced co-factor generated from the PfLDH catalysis. Studies on interference from some common proteins, storage stability, repeatability and analysis of real samples demonstrated the practical application potential of the aptasensor. [ABSTRACT FROM AUTHOR]

المؤلفون: Keersten M. Davis, Christine F. Markwalter, David W. Wright

المصدر: Analytical biochemistry. 493

مصطلحات موضوعية: Immunomagnetic capture, Plasmodium falciparum, Biophysics, 02 engineering and technology, Immunomagnetic separation, 01 natural sciences, Biochemistry, Colorimetry (chemical method), chemistry.chemical_compound, Limit of Detection, Lactate dehydrogenase, parasitic diseases, Humans, Activity assay, Malaria, Falciparum, Molecular Biology, Whole blood, Enzyme Assays, Detection limit, Chromatography, biology, L-Lactate Dehydrogenase, Immunomagnetic Separation, Plasmodium lactate dehydrogenase, 010401 analytical chemistry, Cell Biology, 021001 nanoscience & nanotechnology, biology.organism_classification, Molecular biology, Enzyme assay, Malaria, 0104 chemical sciences, 3. Good health, chemistry, biology.protein, Colorimetry, Antibody, 0210 nano-technology

الوصف: We report a sensitive, magnetic bead-based colorimetric assay for Plasmodium falciparum lactate dehydrogenase (PfLDH) in which the biomarker is extracted from parasitized whole blood and purified based on antigen binding to antibody-functionalized magnetic particles. Antigen-bound particles are washed, and PfLDH activity is measured on-bead using an optimized colorimetric enzyme reaction (limit of detection [LOD] = 21.1 ± 0.4 parasites/μl). Enhanced analytical sensitivity is achieved by removal of PfLDH from the sample matrix before detection and elimination of nonspecific reductases and species that interfere with the optimal detection wavelength for measuring assay development. The optimized assay represents a simple and effective diagnostic strategy for P. falciparum malaria with time-to-result of 45 min and detection limits similar to those of commercial enzyme-linked immunosorbent assay (ELISA) kits, which can take 4–6 h. This method could be expanded to detect all species of malaria by switching the capture antibody on the magnetic particles to a pan-specific Plasmodium LDH antibody.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::0eed1b4d67a48558a5666a947e13ddc1Test
https://pubmed.ncbi.nlm.nih.gov/26475567Test

المؤلفون: Osamu Mori, Yoshimichi Kozuka, Yuji Sakasegawa, Mineo Yamazaki, Takuya Ohkubo, Kiyotoshi Kaneko, Naomi S. Hachiya, Hidehiro Mizusawa

المصدر: Analytical Biochemistry. 347:106-111

مصطلحات موضوعية: Male, Protein Denaturation, Protein Folding, Saccharomyces cerevisiae Proteins, Immunoblotting, Biophysics, Sensitivity and Specificity, Biochemistry, Inclusion bodies, chemistry.chemical_compound, Pick Disease of the Brain, Immunoblot Analysis, Lactate dehydrogenase, Humans, Guanidine, Molecular Biology, Actin, Aged, Inclusion Bodies, L-Lactate Dehydrogenase, Lasers, Cell Biology, chemistry, Unfolded protein response, D-lactate dehydrogenase, Urea, Electrophoresis, Polyacrylamide Gel, Female, L-Lactate Dehydrogenase (Cytochrome), Microdissection

الوصف: We established a histobiochemical approach targeting micron-order inclusion bodies possessing extensive aggregation properties in situ by using a nonchemical denaturant (oligomeric actin interacting protein 2/ d -lactate dehydrogenase protein 2 [Aip2p/Dld2p]) with the combinatorial method of laser-microdissection and immunoblot analysis. As a model, pick bodies were chosen and laser-microdissected from three different brain regions of two patients with Pick’s disease. Initially, 500 to 2000 pick bodies were applied onto SDS–PAGE gels after boiling in Laemmli’s sample buffer according to established immunoblotting procedures; however, only faint signals were obtained. Following negative results with chemical denaturants or detergent, including 6 M guanidine hydrochloride, 8 M urea, and 2% SDS, the laser-microdissected pick bodies were pretreated with oligomeric Aip2p/Dld2p, which possesses robust protein unfolding activity under biological conditions. Strikingly, only one pick body was sufficient to illustrate an immunoblot signal, indicating that pretreatment with oligomeric Aip2p/Dld2p enhanced the immunoblot sensitivity by more than 100-fold. Pretreatment with oligomeric Aip2p/Dld2p also allowed us to quantify the total protein content of pick bodies. Thus, use of oligomeric Aip2p/Dld2p significantly contributed toward the acquisition of information pertaining to the molecular profile of proteins possessing an extensive aggregation property, particularly in small amounts.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::627892b31ee1404df899e21e20fbc9ceTest
https://doi.org/10.1016/j.ab.2005.09.004Test

المؤلفون: Anja-K. Münster-Kühnel, Rita Gerardy-Schahn, Akiko Fujita, Chihiro Sato, Ken Kitajima

المصدر: Analytical Biochemistry. 337:12-21

مصطلحات موضوعية: Cytidine monophosphate, Biophysics, Nicotinamide adenine dinucleotide, Biochemistry, Photometry, Mice, chemistry.chemical_compound, Lactate dehydrogenase, Methods, Animals, Molecular Biology, chemistry.chemical_classification, N-Acylneuraminate Cytidylyltransferase, L-Lactate Dehydrogenase, biology, Cell Biology, NAD, Lyase, Molecular biology, N-Acetylneuraminic Acid, Enzyme assay, Sialic acid, Enzyme, chemistry, biology.protein, NAD+ kinase

الوصف: A new reliable method to assay the activity of cytidine monophosphate sialic acid (CMP-Sia) synthetase (CSS) has been developed. The activation of sialic acids (Sia) to CMP-Sia is a prerequisite for the de novo synthesis of sialoglycoconjugates. In vertebrates, CSS has been cloned from human, mouse, and rainbow trout, and the crystal structure has been resolved for the mouse enzyme. The mouse and rainbow trout enzyme have been compared with respect to substrate specificity, demonstrating that the mouse enzyme exhibits a pronounced specificity for N-acetylneuraminic acid (Neu5Ac), while the rainbow trout CSS is equally active with either of three Sia species, Neu5Ac, N-glycolylneuraminic acid (Neu5Gc), and deaminoneuraminic acid (KDN). However, molecular details that explain the pronounced substrate specificities are unknown. Understanding the catalytic mechanisms of these enzymes is of major importance, since CSSs play crucial roles in cellular sialylation patterns and thus are potential drug targets in a number of pathophysiological situations. The availability of the cDNAs and the obtained structural data enable rational approaches; however, these efforts are limited by the lack of a reliable high-throughput assay system. Here we describe a new assay system that allows product quantification in a reduced nicotinamide adenine dinucleotide (NADH)-dependent color reaction. The activation reaction catalyzed by CSS, CTP+Sia-->CMP-Sia+pyrophosphate, was evaluated by a consumption of Sia, which corresponds to that of NADH on the following two successive reactions: (i) Sia-->pyruvate+ManNAc (or Man), catalyzed by a sialic acid lyase (SAL), and (ii) pyruvate+NADH-->lactate+oxidized nicotinamide adenine dinucleotide (NAD+), catalyzed by a lactate dehydrogenase (LDH). Consumption of NADH can be photometrically monitored on a microtiter plate reader for a number of test samples at the same time. Furthermore, based on the quantification of CSS used in the SAL/LDH assay, relative activities toward Sia derivatives have been obtained. The preference of mouse CSS toward Neu5Ac and the ability of the rainbow trout enzyme to activate both KDN and Neu5Ac were confirmed. Thus, this simple and time-saving method is suitable for a systematic comparison of enzyme activity of structurally mutated enzymes based on the relative specific activity.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::902e844971ce864bac25fed84c85b12cTest
https://doi.org/10.1016/j.ab.2004.10.033Test

المؤلفون: Maxim Zakhartsev, Hans O. Pörtner, Ronny Blust

المصدر: Analytical biochemistry
EPIC3Analytical Biochemistry, 330, pp. 10-20

مصطلحات موضوعية: 030310 physiology, Biophysics, Analytical chemistry, Activation energy, Biochemistry, 03 medical and health sciences, chemistry.chemical_compound, symbols.namesake, Lactate dehydrogenase, Spectrophotometry, medicine, Animals, Gadus, Muscle, Skeletal, Molecular Biology, 030304 developmental biology, Arrhenius equation, 0303 health sciences, Temperature control, L-Lactate Dehydrogenase, biology, medicine.diagnostic_test, Cell Biology, biology.organism_classification, Microplate Reader, Freezing point, Cold Temperature, Kinetics, Gadus morhua, chemistry, symbols, Thermodynamics

الوصف: Analyses of temperature-dependent kinetic parameters in enzymes extracted from tissues of ectothermic animals are usually carried out within the range of physiological temperatures (0-40 degrees C). However, multisample spectrophotometers (so-called microplate readers) with efficient wide-range temperature control (including cooling) have previously been unavailable. This limits the statistical quality of the measurements. A temperature-controlled microplate was designed for a 96-well microplate reader to overcome this limitation. This so-called T-microplate is able to control assay temperature between the freezing point of a liquid sample and 60 degrees C with high stability and accuracy in any data acquisition mode. At 4 degrees C the accuracy of the temperature control was +/-0.1 degrees C and temperature homogeneity across the microplate was +/-0.3 degrees C. As examples, analyses of the temperature dependence of Michaelis-Menten (K'(PYR)(m) and substrate inhibition (K'(PYR)(si) constants for pyruvate, of the maximal rate of reaction (V'(max), of the apparent Arrhenius activation energy (E(A), and of the Gibbs free-energy change (deltaG) of lactate dehydrogenases from muscle of Atlantic cod Gadus morhua acclimated to 4 degrees C are described. The large dataset obtained allowed evaluation of a new mechanism of metabolic compensation in response to seasonal temperature change.

وصف الملف: application/pdf

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::f879837c6a1e331b058984fed0e3818aTest
https://doi.org/10.1016/j.ab.2004.03.070Test

المؤلفون: Catherine J. Pachuk, Meiqing Lu, Manoj Samuel, Chandrasekha Satishchandran

المصدر: Analytical Biochemistry. 313:301-306

مصطلحات موضوعية: DNA, Bacterial, Pyruvate Kinase, Biophysics, Sensitivity and Specificity, Biochemistry, Substrate Specificity, chemistry.chemical_compound, Adenosine Triphosphate, RNA, Transfer, Lactate dehydrogenase, Escherichia coli, Magnesium, Molecular Biology, chemistry.chemical_classification, Deoxyribonucleases, Chromatography, DNA clamp, L-Lactate Dehydrogenase, Hydrolysis, Substrate (chemistry), Deoxyribonuclease, Cell Biology, NAD, Adenosine Diphosphate, Kinetics, Enzyme, chemistry, Spectrophotometry, Phosphodiester bond, NAD+ kinase, DNA, Circular, Oxidation-Reduction, DNA, Plasmids

الوصف: A spectrophotometric method for quantification of linear DNA is described. The assay measures ADP produced following digestion of linear DNA by an ATP-dependent deoxyribonuclease. Cleavage of the phosphodiester bond of the DNA substrate is proportional to ADP formed in the reaction which follows typical Michaelis-Menten kinetics (K(m) of 0.6 microM, and a V(max) of 30 nmol/min/mg). The enzyme requires Mg(2+)-ATP and Mg(2+)-DNA as substrates, although the results suggest a requirement for yet another metal ion which may be enzyme bound. Both single-stranded and double-stranded linear DNA are substrates, as demonstrated by comparable initial velocity measurements. However, covalently closed circular (CCC) and nicked open circular DNA are not substrates for the enzyme. The rate of hydrolysis of ATP is not inhibited by 1 microg RNA or covalently closed circular DNA. The product (ADP) formed in the reaction is coupled to NADH oxidation using pyruvate kinase and lactate dehydrogenase. NAD formed in the reaction is monitored spectrophotometrically as a loss in absorbance at 340 nm. This assay directly measures the amount of linear DNA present in preparations of supercoiled (CCC) plasmid DNA, and has direct utility for monitoring the quality of plasmid preparations for gene therapy.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::02f1b85041de42dc2d5abb4a2612d293Test
https://doi.org/10.1016/s0003-2697Test(02)00591-2

المؤلفون: Ben de Kruijff, Marjolein J. F. W. Janssen, Anton I.P.M. de Kroon

المصدر: Analytical Biochemistry. 300:27-33

مصطلحات موضوعية: Saccharomyces cerevisiae Proteins, Mitochondrial intermembrane space, Biophysics, Saccharomyces cerevisiae, Biology, Mitochondrial Membrane Transport Proteins, Biochemistry, Permeability, Membrane Potentials, Phosphates, Mitochondrial membrane transport protein, Oxygen Consumption, Magnesium, Trypsin, Inner mitochondrial membrane, Molecular Biology, Fluorescent Dyes, Membrane potential, L-Lactate Dehydrogenase, Chemiosmosis, Membrane Transport Proteins, Intracellular Membranes, Cell Biology, Mitochondria, Cell biology, Spectrometry, Fluorescence, Translocase of the inner membrane, biology.protein, L-Lactate Dehydrogenase (Cytochrome), Energy Metabolism, Bacterial outer membrane, Intermembrane space

الوصف: The buffer requirements to maintain mitochondrial intactness and membrane potential in in vitro studies were investigated, using gradient purified yeast mitochondria. It was found that the presence of phosphate is crucial for generation of a stable membrane potential and for preserving the intactness of the outer membrane, as assessed by probing the accessibility of Tom40p to trypsin and the leakage of cytochrome b2 from the intermembrane space. Upon addition of respiratory substrate in the absence of phosphate, mitochondria generate a membrane potential that collapses within 1 min. Under the same conditions, the mitochondrial outer membrane is disrupted. The presence of phosphate prevents both phenomena. The DeltapH component of the proton motive force appears to be responsible for the compromised outer membrane integrity. The collapse of the membrane potential is reversible to a limited extent. Only when phosphate is added soon enough after the addition of exogenous respiratory substrate can a stable membrane potential be obtained again. Within a few minutes, this capacity is lost. The presence of Mg(2+) prevents rupture of the outer membrane, but does not prevent rapid dissipation of the membrane potential. Similar results were obtained for mitochondria isolated and stored in the presence of dextran or bovine serum albumin.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::428d1d211d44ed5472c224d675788416Test
https://doi.org/10.1006/abio.2001.5438Test

المؤلفون: Fokou, Patrick Valere Tsouh^1,2 (AUTHOR), Tali, Brice Mariscal Tchatat¹ (AUTHOR), Dize, Darline¹ (AUTHOR), Mbouna, Cedric Derick Jiatsa¹ (AUTHOR), Ngansop, Cyrille Armel Njampa¹ (AUTHOR), Keumoe, Rodrigue¹ (AUTHOR), Tchokouaha, Lauve Rachel Yamthe^1,3 (AUTHOR), Tchouankeu, Jean Claude⁴ (AUTHOR), Escudie, Fanny⁵ (AUTHOR), Duffy, James⁵ (AUTHOR), Boyom, Fabrice Fekam¹ (AUTHOR) fabrice.boyom@fulbrightmail.org

المصدر: Analytical Biochemistry. Jul2022, Vol. 648, pN.PAG-N.PAG. 1p.

مصطلحات موضوعية: *MALARIA, *PLASMODIUM falciparum, *LACTATE dehydrogenase, *MIDDLE-income countries, *HIGH throughput screening (Drug development), *LACTATES

مستخلص: Antimalarial drug discovery has been facilitated by the development of various in vitro drug susceptibility testing methods suitable for medium-throughput or high-throughput campaigns. Among many, the Plasmodium falciparum lactate dehydrogenase (Pf LDH) assay has acceptable demand on equipment, labour, technical skills and affordability and offers a good opportunity for scientists in low- and middle-income countries to participate in the global effort of discovering future antimalarial drugs. Hence, to enable our search for novel antimalarial drugs, we implemented and examined assay conditions and validated the Pf LDH-based method in our laboratory using a reference set of standard antimalarial drugs with known activity against Plasmodium falciparum strains. The Pf LDH assay revealed acceptable linearity profiles of R2 = 0.97 and 0.92 for Pf 3D7 and Pf Dd2, respectively, achieved at 2% parasitaemia and 1% haematocrit. The detection and quantitation limits (DL and QL) of the Pf LDH-based assay were 0.09% and 0.4% parasitemia, respectively. The assay showed an acceptable average Z-factor between 0.76 and 0.79 and was considerably robust. The average interassay reproducibility via percent coefficient of variation (%CV) was 5.47 between independent experiments. Overall, the Pf LDH-based method produced a reliable and reproducible drug screening profile for in vitro assays in our setting. There were no significant interassay variability or hazards of other screening assays. [Display omitted] • Pf LDH-based method was implemented and assay conditions examined and validated. • The Pf LDH assay revealed acceptable linearity profiles. • The assay showed acceptable Z-factor range between 0.76 and 0.79. • Pf LDH-based method was reliable and reproducible for in vitro drug testing. [ABSTRACT FROM AUTHOR]

المؤلفون: Pranab Goswami, Babina Chakma, Priyamvada Jain, Smita Das

المصدر: Analytical biochemistry. 514

مصطلحات موضوعية: Aptamer, Plasmodium falciparum, Biophysics, Analytical chemistry, Oxide, 02 engineering and technology, Biosensing Techniques, 010402 general chemistry, Electrochemistry, 01 natural sciences, Biochemistry, Sensitivity and Specificity, law.invention, symbols.namesake, chemistry.chemical_compound, law, Humans, Graphite, Molecular Biology, Electrodes, L-Lactate Dehydrogenase, Graphene, Chemistry, Reproducibility of Results, Oxides, Cell Biology, Electrochemical Techniques, Equipment Design, Aptamers, Nucleotide, 021001 nanoscience & nanotechnology, 0104 chemical sciences, Electrode, symbols, 0210 nano-technology, Raman spectroscopy, Systematic evolution of ligands by exponential enrichment, Nuclear chemistry

الوصف: A 90 mer ssDNA aptamer (P38) enriched against Plasmodium falciparum lactate dehydrogenase (PfLDH) through SELEX process was immobilized over glassy carbon electrode (GCE) using graphene oxide (GO) as an immobilization matrix, and the modified electrode was investigated for detection of PfLDH. The GO was synthesized from powdered pencil graphite and characterized by XRD based on the increased interlayer distance between graphitic layers from 0.345 nm for graphite to 0.829 nm for GO. The immobilization of P38 on GO was confirmed by ID/IG intensity ratio in Raman spectra where, the ratio were 0.67, 0.915, and 1.35 for graphite, GO and P38-GO, respectively. The formation of the P38 layer over GO-GCE was evident from an increase in the surface height in AFM analysis of the electrode from ∼3.5 nm for GO-GCE to ∼27 nm for P38-GO-GCE. The developed aptasensor when challenged with the target, a detection of as low as 0.5 fM of PfLDH was demonstrated. The specificity of the aptasensor was confirmed through a voltametric measurement at 0.65 V of the reduced co-factor generated from the PfLDH catalysis. Studies on interference from some common proteins, storage stability, repeatability and analysis of real samples demonstrated the practical application potential of the aptasensor.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::7c51a6a0bbee611c3627d2242e8fed12Test
https://pubmed.ncbi.nlm.nih.gov/27641111Test

المؤلفون: Gareth W. Morgan, Catherine M. Halliwell, Anthony E. G. Cass, Chung-Pei Ou

المصدر: Analytical Biochemistry. 295:257-261

مصطلحات موضوعية: chemistry.chemical_classification, Protein Folding, L-Lactate Dehydrogenase, Chemistry, Kinetics, Biophysics, Dehydrogenase, L-Lactate dehydrogenase, Cell Biology, Biochemistry, Chromatography, Affinity, Geobacillus stearothermophilus, Enzyme, Affinity chromatography, Chromatography, Gel, Molecule, Histidine, Protein folding, Molecular Biology

الوصف: A (poly)histidine tag was fused to either the N- or the C-terminus of L-lactate dehydrogenase (LDH) of Bacillus stearothermophilus to facilitate purification and immobilization of these enzymes. The C-terminally tagged enzyme displayed lower activity compared both to the wild-type and to the N-terminally tagged variant. The reason for this loss of activity was investigated by affinity chromatography of the enzymes on a 5'-AMP-Sepharose resin and by size-exclusion chromatography. The C-terminally tagged enzyme could be separated into an inactive, unbound fraction and an active, bound fraction. Further differences between the C-terminally tagged enzyme and the N-terminally tagged and wild-type LDH were observed on size-exclusion chromatography of the three enzymes. These data suggest that the introduction of a "his-tag" at the C-terminus may induce misfolding of the LDH and serve as a warning that the introduction of a (poly)histidine tag can produce unforseen changes in a protein.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::7fea8e0180eb96e9e720167bcae32513Test
https://doi.org/10.1006/abio.2001.5182Test