scholarly journals KD determination from time-resolved experiments on live cells with LigandTracer and reconciliation with end-point flow cytometry measurements

2021 ◽  
Author(s):  
Diana Spiegelberg ◽  
Jonas Stenberg ◽  
Pascale Richalet ◽  
Marc Vanhove
Keyword(s):  
Flow Cytometry ◽  
Live Cells ◽  
Time Resolved ◽  
Surface Receptors ◽  
Full Characterization ◽  
End Point ◽  
Titration Curves ◽  
Kinetics Of

AbstractDesign of next-generation therapeutics comes with new challenges and emulates technology and methods to meet them. Characterizing the binding of either natural ligands or therapeutic proteins to cell-surface receptors, for which relevant recombinant versions may not exist, represents one of these challenges. Here we report the characterization of the interaction of five different antibody therapeutics (Trastuzumab, Rituximab, Panitumumab, Pertuzumab, and Cetuximab) with their cognate target receptors using LigandTracer. The method offers the advantage of being performed on live cells, alleviating the need for a recombinant source of the receptor. Furthermore, time-resolved measurements, in addition to allowing the determination of the affinity of the studied drug to its target, give access to the binding kinetics thereby providing a full characterization of the system. In this study, we also compared time-resolved LigandTracer data with end-point KD determination from flow cytometry experiments and hypothesize that discrepancies between these two approaches, when they exist, generally come from flow cytometry titration curves being acquired prior to full equilibration of the system. Our data, however, show that knowledge of the kinetics of the interaction allows to reconcile the data obtained by flow cytometry and LigandTracer and demonstrate the complementarity of these two methods.

Kinetics of lysozyme refolding: structural characterization of a non-specifically collapsed state using time-resolved X-ray scattering

1998 ◽  
Vol 276 (1) ◽  
pp. 225-237 ◽  
Author(s):  
Lingling Chen ◽  
Gudrun Wildegger ◽  
Thomas Kiefhaber ◽  
Keith O Hodgson ◽  
Sebastian Doniach
Keyword(s):  
Structural Characterization ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Scattering ◽  
Lysozyme Refolding ◽  
Kinetics Of ◽  
Ray Scattering

Hydrogen Passivation Kinetics of Si Nanocrystals in SiO2

2003 ◽  
Vol 770 ◽  
Author(s):  
Andrew R. Wilkinson ◽  
Robert G. Elliman
Keyword(s):  
Reaction Kinetics ◽  
Activation Energies ◽  
Hydrogen Passivation ◽  
Time Resolved ◽  
Luminescence Efficiency ◽  
Si Nanocrystals ◽  
Preliminary Study ◽  
Kinetics Of ◽  
Insight Into

AbstractHydrogen passivation of non-radiative defects increases the luminescence intensity from silicon nanocrystals. In this study, photoluminescence (PL) and time-resolved PL were used to investigate the chemical kinetics of the hydrogen passivation process. Isochronal and isothermal annealing sequences were used to determine the reaction kinetics for the absorption and desorption of hydrogen, using the generalised consistent simple thermal (GST) model proposed by Stesmans for Pb defects at planar Si/SiO2 interfaces. This included determination of the activation energies and rate constants for the forward and reverse reactions as well as the associated spread in activation energies. The reaction kinetics determined from such measurements were found to be in excellent agreement with those for the passivation of Pb defects at planar Si/SiO2 interfaces, suggesting the nanocrystal emission process is also limited by such defects. These results provide useful model data as well as insight into the processing conditions needed to achieve optimum passivation in H2. As an extension to the work, a preliminary study into passivation by atomic hydrogen was pursued via a post-metallization Al anneal (alneal). A considerable gain in luminescence efficiency was achieved over the previously optimised passivation in H2.

scholarly journals Application of flow cytometry and cell sorting to megakaryocytopoiesis

Blood ◽  
1979 ◽  
Vol 53 (4) ◽  
pp. 732-745 ◽  
Author(s):  
A Nakeff ◽  
F Valeriote ◽  
JW Gray ◽  
RJ Grabske
Keyword(s):  
Flow Cytometry ◽  
Cell Sorting ◽  
Significant Advance ◽  
Glass Slides ◽  
Acetylcholinesterase Staining ◽  
Fcm Analysis ◽  
Cell Fractions ◽  
Kinetics Of ◽  
Supravital Staining

Abstract We have employed flow cytometry (FCM) and cell sorting to quantitate and study megakaryocytes in mouse and rat femoral marrow following their 20- to 30-fold concentration by centrifugal elutriation (CE). This enrichment of megakaryocytes permitted the first determination of their DNA-related fluorescence by FCM analysis following DNA staining. Fluorescence distributions of CE-enriched cell fractions following supravital staining with Hoechst 33342 were similar to those following chromomycin A3 staining of ethanol-fixed cells. Microscopic examination of cells sorted onto glass slides on the basis of their DNA-related fluorescence following supravital staining together with specific acetylcholinesterase staining for megakaryocytes indicated that megakaryocytes generally increased in cell size with increasing DNA content. This technologic application represents a significant advance in the study of megakaryocytopoiesis, since the kinetics of either the normal or perturbed population can now be studied rapidly and quantitatively.

scholarly journals Kinetic and substrate complex characterization of RamA, a corrinoid protein reductive activase from Methanosarcina barkeri

2020 ◽  
Vol 367 (17) ◽  
Author(s):  
Katherine A Huening ◽  
Ruisheng Jiang ◽  
Joseph A Krzycki
Keyword(s):  
Direct Evidence ◽  
Atp Hydrolysis ◽  
Methanogenic Archaea ◽  
Methanosarcina Barkeri ◽  
Serine Residue ◽  
Substrate Complex ◽  
Apparent Kinetic Parameters ◽  
Kinetics Of

ABSTRACT In microbial corrinoid-dependent methyltransferase systems, adventitious Co(I)-corrinoid oxidation halts catalysis and necessitates repair by ATP-dependent reductive activases. RamA, an activase with a C-terminal ferredoxin domain with two [4Fe-4S] clusters from methanogenic archaea, has been far less studied than the bacterial activases bearing an N-terminal ferredoxin domain with one [2Fe-2S] cluster. These differences suggest RamA might prove to have other distinctive characteristics. Here, we examine RamA kinetics and the stoichiometry of the corrinoid protein:RamA complex. Like bacterial activases, K+ stimulates RamA. Potassium stimulation had been questioned due to differences in the primary structure of bacterial and methanogen activases. Unlike one bacterial activase, ATP is not inhibitory allowing the first determination of apparent kinetic parameters for any corrinoid activase. Unlike bacterial activases, a single RamA monomer complexes a single corrinoid protein monomer. Alanine replacement of a RamA serine residue corresponding to the serine of one bacterial activase which ligates the corrinoid cobalt during complex formation led to only moderate changes in the kinetics of RamA. These results reveal new differences in the two types of corrinoid activases, and provide direct evidence for the proposal that corrinoid activases act as catalytic monomers, unlike other enzymes that couple ATP hydrolysis to difficult reductions.

scholarly journals Characterization of the CYP3A4 Enzyme Inhibition Potential of Selected Flavonoids

Molecules ◽  
2021 ◽  
Vol 26 (10) ◽  
pp. 3018
Author(s):  
Martin Kondža ◽  
Mirza Bojić ◽  
Ivona Tomić ◽  
Željan Maleš ◽  
Valentina Rezić ◽  
...  
Keyword(s):  
Covalent Binding ◽  
Inhibitory Effect ◽  
Residual Concentration ◽  
Flavonoid Aglycones ◽  
Irreversible Inhibitors ◽  
Cyp3a4 Enzyme ◽  
Kinetics Of ◽  
Kinetics Of Inhibition

Acacetin, apigenin, chrysin, and pinocembrin are flavonoid aglycones found in foods such as parsley, honey, celery, and chamomile tea. Flavonoids can act as substrates and inhibitors of the CYP3A4 enzyme, a heme containing enzyme responsible for the metabolism of one third of drugs on the market. The aim of this study was to investigate the inhibitory effect of selected flavonoids on the CYP3A4 enzyme, the kinetics of inhibition, the possible covalent binding of the inhibitor to the enzyme, and whether flavonoids can act as pseudo-irreversible inhibitors. For the determination of inhibition kinetics, nifedipine oxidation was used as a marker reaction. A hemochromopyridine test was used to assess the possible covalent binding to the heme, and incubation with dialysis was used in order to assess the reversibility of the inhibition. All the tested flavonoids inhibited the CYP3A4 enzyme activity. Chrysin was the most potent inhibitor: IC50 = 2.5 ± 0.6 µM, Ki = 2.4 ± 1.0 µM, kinact = 0.07 ± 0.01 min−1, kinact/Ki = 0.03 min−1 µM−1. Chrysin caused the highest reduction of heme (94.5 ± 0.5% residual concentration). None of the tested flavonoids showed pseudo-irreversible inhibition. Although the inactivation of the CYP3A4 enzyme is caused by interaction with heme, inhibitor-heme adducts could not be trapped. These results indicate that flavonoids have the potential to inhibit the CYP3A4 enzyme and interact with other drugs and medications. However, possible food–drug interactions have to be assessed clinically.

scholarly journals Effect of the Exchanged Cation in an Algerian Montmorillonite Used as a Heterogeneous Catalyst for Biginelli Reaction

2021 ◽  
Vol 68 (2) ◽  
pp. 355-362
Author(s):  
Fatiha Belferdi ◽  
Farida Bouremmad ◽  
Shalima Shawuti ◽  
Mehmet Ali Gulgun
Keyword(s):  
Heterogeneous Catalyst ◽  
Catalytic Properties ◽  
Biginelli Reaction ◽  
Reaction Medium ◽  
Exchange Capacity ◽  
Basal Spacing ◽  
Exchange Operation ◽  
Kinetics Of

In this work, an Algerian montmorillonite (Mt) is exchanged by different cations from the transition metals family, namely: Cu2+, Ni2+, Cr3+, Co2+, Fe2+ and Fe3+, it is used as a heterogeneous catalyst for Biginelli reaction. The exchanged cations are known for their catalytic properties in homogeneous catalysis. The main purpose is to study the effect of the exchanged cations on the yield and the kinetics of the reaction. The characterization of montmorillonite was carried out by XRD, which allows us to follow the evolution of the basal spacing d001 as a function of the exchanged cation and to show that the exchange operation has not altered the montmorillonite structure. The cation exchange capacity (CEC) is determined by the titration of the exchanged cation by atomic absorption. The product of the reaction is characterized by NMR, IR and by the determination of the melting point. In addition, the importance of the introduction order of the reagents into the reaction medium has been demonstrated on the yield and the kinetics. Finally, the obtained results show that the exchanged montmorillonite is competitive with other costly heterogeneous and homogeneous catalysts.

scholarly journals A novel assay based on pre-equilibrium titration curves for the determination of enzyme inhibitor binding kinetics

2021 ◽  
Author(s):  
Bernard Noppen ◽  
Anouk Vanbelle ◽  
Alan W. Stitt ◽  
Marc Vanhove
Keyword(s):  
Enzyme Inhibitors ◽  
Enzyme Inhibitor ◽  
Peptide Inhibitors ◽  
Pharmacological Agents ◽  
Enzymatic Assays ◽  
Target Interaction ◽  
Titration Curves ◽  
Selection Of

AbstractSelection of pharmacological agents based on potency measurements performed at equilibrium fail to incorporate the kinetic aspects of the drug–target interaction. Here we describe a method for screening or characterization of enzyme inhibitors that allows the concomitant determination of the equilibrium inhibition constant in unison with rates of complex formation and dissociation. The assay is distinct from conventional enzymatic assays and is based on the analysis of inhibition curves recorded prior to full equilibration of the system. The methodology is illustrated using bicyclic peptide inhibitors of the serine protease plasma kallikrein.

scholarly journals Glycoscience@Synchrotron: Synchrotron radiation applied to structural glycoscience

2017 ◽  
Vol 13 ◽  
pp. 1145-1167 ◽  
Author(s):  
Serge Pérez ◽  
Daniele de Sanctis
Keyword(s):  
Synchrotron Radiation ◽  
Structural Characterization ◽  
Crystalline State ◽  
Structural Diversity ◽  
Interacting Proteins ◽  
X Ray ◽  
Wide Range ◽  
Kinetics Of

Synchrotron radiation is the most versatile way to explore biological materials in different states: monocrystalline, polycrystalline, solution, colloids and multiscale architectures. Steady improvements in instrumentation have made synchrotrons the most flexible intense X-ray source. The wide range of applications of synchrotron radiation is commensurate with the structural diversity and complexity of the molecules and macromolecules that form the collection of substrates investigated by glycoscience. The present review illustrates how synchrotron-based experiments have contributed to our understanding in the field of structural glycobiology. Structural characterization of protein–carbohydrate interactions of the families of most glycan-interacting proteins (including glycosyl transferases and hydrolases, lectins, antibodies and GAG-binding proteins) are presented. Examples concerned with glycolipids and colloids are also covered as well as some dealing with the structures and multiscale architectures of polysaccharides. Insights into the kinetics of catalytic events observed in the crystalline state are also presented as well as some aspects of structure determination of protein in solution.

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