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 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.

scholarly journals Enzymic characterization with progress curve analysis of a collagen peptidase from an enthomopathogenic bacterium, Photorhabdus luminescens

2004 ◽  
Vol 379 (3) ◽  
pp. 633-640 ◽  
Author(s):  
Judit MAROKHÁZI ◽  
György KÓCZÁN ◽  
Ferenc HUDECZ ◽  
László GRÁF ◽  
András FODOR ◽  
...  
Keyword(s):  
Enzyme Inhibitors ◽  
Integration Method ◽  
Significant Activity ◽  
Photorhabdus Luminescens ◽  
Ph Optimum ◽  
Collagen Peptide ◽  
Progress Curve ◽  
Bivalent Metal Ions

A proteolytic enzyme, Php-B (Photorhabdus protease B), was purified from the entomopathogenic bacterium, Photorhabdus luminescens. The enzyme is intracellular, and its molecular mass is 74 kDa. Tested on various peptide and oligopeptide substrates, Php-B hydrolysed only oligopeptides, with significant activity against bradykinin and a 2-furylacryloyl-blocked peptide, Fua-LGPA (2-furylacryloyl-Leu-Gly-Pro-Ala; kcat=3.6×102 s−1, Km=5.8×10−5 M−1, pH optimum approx. 7.0). The pKa1 and the pKa2 values of the enzyme activity (6.1 and 7.9 respectively), as well as experiments with enzyme inhibitors and bivalent metal ions, suggest that the activity of Php-B is dependent on histidine and cysteine residues, but not on serine residues, and that it is a metalloprotease, which most probably uses Zn2+ as a catalytic ion. The enzyme's ability to cleave oligopeptides that contain a sequence similar to collagen repeat (-Pro-Xaa-Gly-), bradykinin and Fua-LGPA (a synthetic substrate for bacterial collagenases and collagen peptidases), but not native collagens (types I and IV) or denatured collagen (gelatin), indicates that Php-B is probably a collagen peptidase, the first enzyme of this type to be identified in an insect pathogen, that might have a role in the nutrition of P. luminescens by degrading small collagen fragments. For the determination of enzyme kinetic constants, we fitted a numerically integrated Michaelis–Menten model to the experimental progress curves. Since this approach has not been used before in the characterization of proteases that are specific for the P1´–P4´ substrate sites (e.g. collagenolytic enzymes), we present a comparison of this method with more conventional ones. The results confirm the reliability of the numerical integration method in the kinetic analysis of collagen-peptide-hydrolysing enzymes.

scholarly journals NATURAL CATHEPSIN INHIBITOR TO INHIBIT MILKFISH DETERIORATION DURING CHILLING STORAGE

2016 ◽  
Vol 7 (2) ◽  
Author(s):  
Tati Nurhayati ◽  
Wini Trilaksani ◽  
Mohammad Zaenuri
Keyword(s):  
Enzyme Inhibitors ◽  
Enzyme Inhibitor ◽  
Relative Activity ◽  
Observation Time ◽  
Time Interval ◽  
Extraction Temperature ◽  
Rigor Mortis ◽  
Fish Quality ◽  
Quality Deterioration

Quality deterioration of fish is caused by various factors, one of them is due to the activity of cathepsin enzyme. Cathepsin enzyme activity can be inhibited by enzyme inhibitors. Inhibitors of the enzyme can be obtained from fish meat tissue by separating the enzyme-inhibitor complex with heat treatment. The purpose of this research was to extract and apply the cathepsin inhibitors to inhibit deterioration of fish quality. This research was conducted in three  steps: (1) the determination of the optimum extraction temperature for extraction of cathepsin inhibitors, (2) the determination of the most potent cathepsin inhibitors with various concentrations, and (3) the application of cathepsin inhibitors in inhibiting the deterioration of fish quality. The results showed that the cathepsin inhibitors was optimally extracted at 80°C. Cathepsin inhibitors diluted using buffer with a ratio of 1: 1 had the same relative activity without dilution. The process of quality deterioration of milkfish  control in chilling temperatures occured after 504 hours (21 days) storage with a 24-hour observation time interval. The complete process during observation was pre-rigor (0 hour ), rigor mortis (96 hours), post-rigor (360 hours), and deterioration (504 hours). The process of milkfish quality deterioration treated with soaking in  cathepsin inhibitors occured after 624 hours (26 days) storage with the detailed process as follows: 0 hour (pre-rigor), 144 hours (rigor mortis), 408 hours (post-rigor), and 624 hours (deterioration). Thus fish marinated with cathepsin inhibitors achieve slower deterioration phase 5 days. Keywords: cathepsin inhibitor, deterioration, pre-rigor, rigor mortis, post-rigor, quality deterioration.

Stray light and ghosts in catadioptric spectrometers: incorporating grating scatter measurements into simulations and ghost sensitivity into system design

2018 ◽  
Vol 7 (6) ◽  
pp. 377-386 ◽  
Author(s):  
Monika Kroneberger ◽  
Andreas Mezger ◽  
Stephanie Becker
Keyword(s):  
High Sensitivity ◽  
Stray Light ◽  
Multiple Diffraction ◽  
Optical Elements ◽  
Optical Instruments ◽  
Light Requirements ◽  
Measurement Results ◽  
Selection Of

Abstract The accurate simulation of stray light is essential for the verification of the contrast requirements in optical instruments. In a spectrometer, the scattering from reflective gratings is difficult to characterize while contributing significantly to the overall system stray light and reduction of the spectrometer contrast. In addition, the multiple diffraction orders create a ghost sensitive environment, which must be considered in the design of the instrument. In this article, we present an experimental setup for, and measurement results from, the characterization of the bidirectional scattering distribution function (BSDF) of a holographic grating for a spectrometer applied in a typical earth observation mission with demanding stray light requirements. We observed distinct stray light peaks out of the diffraction plane, which are called ‘satellites.’ The main challenges in the measurement of grating BSDFs arise from the near angle limit, the determination of the instrument signature and the selection of the appropriate sampling (2D or 3D). Following the grating characterization, the next step is to introduce these measured BSDFs into stray light simulation. We have done that by fitting appropriate functions to the measured BSDF and defining them in the optical analysis software ASAP as a user-defined BSDF. Ghost analysis is done at the spectrometer level as a sensitivity analysis of the tilts of the optical elements. Due to the ghosting of higher diffraction orders of the grating, a high sensitivity to the tilts of some of the optical elements can be seen.

Wavelet Based Separation for Synthetic Topographical Characterization of Surface Prepared by Abrasive Water Jet

2012 ◽  
Vol 229-231 ◽  
pp. 2648-2652 ◽  
Author(s):  
Song Lin Wu ◽  
J. Valicek
Keyword(s):  
Wavelet Decomposition ◽  
Water Jet ◽  
Abrasive Water Jet ◽  
Optical Instrument ◽  
Form Error ◽  
Decomposition Level ◽  
Wavelet Bases ◽  
Selection Of

To study the synthetic topographical characterization of surface created by abrasive water jet (AWJ) and carry out a detailed research for the process of surface creating, this paper researches the surface signals obtained both by contact profiler and a new developed optical instrument. Through wavelet decomposition and re-construction of surface signal, surface features, such as form error, waviness and roughness are separated rationally. Some fundamental issues, such as the selection of wavelet bases and the determination of wavelet decomposition level are discussed in detail.

scholarly journals NON-DESTRUCTIVE METHOD FOR RAPID "IN SITU" CHARACTERIZATION OF ROCKS

2004 ◽  
Vol 36 (4) ◽  
pp. 1912
Author(s):  
N. Ciornei ◽  
I. Facaoaru ◽  
V. Cetean
Keyword(s):  
Quality Criteria ◽  
In Situ Characterization ◽  
Hard Rocks ◽  
Determination Process ◽  
Building Activity ◽  
Non Destructive ◽  
Selection Of

The effective determination of the field and place of application of the products resulting from stone working is a compulsory requirement in the building activity and is solved by laboratory analyses. This article presents details on the determination, by non-destructive method, of the compression strength of the rocks, both "in situ" (in the deposit) and in a laboratory. The originality of the method consists in simplifying the determination process of this parameter, a method that is in agreement with the systems proposed to be applied in the European Community. The method has the advantage of being applicable in quarries, where knowledge of this parameter leads to a rationalizing of the working and more especially to a selection of the blocks by quality criteria. The method is applicable to all categories of natural consolidated hard rocks, being a viable alternative for deposits with an inhomogeneous composition as to quality and structure.

scholarly journals Molecular and Elemental Characterization of Selected Turkish Durum Wheat Varieties

2014 ◽  
Vol 42 (2) ◽  
pp. 431-439
Author(s):  
Erdogan E. HAKKI ◽  
Nurdan DOGRAR ◽  
Anamika PANDEY ◽  
Mohd. Kamran KHAN ◽  
Mehmet HAMURCU ◽  
...  
Keyword(s):  
Microsatellite Markers ◽  
Durum Wheat ◽  
Molecular Data ◽  
Wheat Varieties ◽  
Elemental Analyses ◽  
Principle Coordinate Analysis ◽  
Elemental Characterization ◽  
Selection Of

Combination of elemental and protein studies along with molecular data using microsatellite markers may lead to the better and realistic determination of relatedness between the varieties and their populations. In this study, the extent of diversity among five Turkish durum wheat cultivars and their populations has been assessed using seven microsatellite markers and the elemental analysis together with the differences in their protein content.In molecular analysis, total 23 alleles have been obtained among all the genotypes with middling of 4.6 per primer. On employing UPGMA Dendrogram, Principle Coordinate Analysis (PCoA) and Winboot analyses, both inter and intra varietal polymorphic studies had shown similar clustering with minor differences. As a result of AMOVA performed, the extent of diversity was found to be higher among the genotypes (76%) in comparison to the variability within the genotypes (24%). In elemental analyses, ‘Selcuklu-97’ was found to be the most efficient variety with high content of several elements. Also, strong and positive correlation has been observed between magnesium- phosphorus, magnesium-sulphur and sulphur-sodium, while noteworthy negative correlation has been observed between sodium and zinc. The protein content of the genotypes was found in the range of 15.17-16.90%.The diversity revealed in durum genotypes can be employed in genetic expansion of the crop. These involved varieties may aid to avoid genetic attrition coming up from the landraces. The information provided can be utilized by breeders for appropriate selection of both, genetically and nutritionally efficient durum wheat varieties.

scholarly journals The Beginning of Western Greek Amphorae Production in Western Sicily: Archaeometric and Archaeological Studies on 6th–5th Centuries BCE Amphorae Manufactured in Himera

Minerals ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 762
Author(s):  
Giuseppe Montana ◽  
Luciana Randazzo ◽  
Babette Bechtold
Keyword(s):  
Special Focus ◽  
Data Set ◽  
Reflected Light ◽  
Icp Ms ◽  
Economic Interaction ◽  
Western Sicily ◽  
Thin Section Petrography ◽  
Selection Of

About 560 western Greek amphorae (6th–5th centuries BCE) re-used in enchytrismos burials were unearthed in the necropolis of the Dorian-Chalcidian colony of Himera in northwestern Sicily. Among the most striking issues is the determination of their geographical provenance. For this purpose, ceramic samples chipped from freshly broken surfaces of all the amphorae were first subdivided into macrofabrics by the use of a hand lens. Thereon, the samples were studied in accordance with standardized methods by the use of reflected light microscopy. Due to the special focus of our project on the characterization of Sicilian productions, a selection of amphorae which showed visible, macroscopic affinities with the majority of the macrofabrics previously attributed to the region of Himera was submitted for thin-section petrography at the polarizing microscope and chemical analyses (ICP-MS and ICP/OES). This new data set was compared with reference samples investigated by previous research, referring to ceramic raw clays of the colony’s territory and local tablewares of the Iato K480-type. Our study confirms the local manufacture of the entire selection of transport vessels. The identification of a production of western Greek wine (?) amphorae in Himera dating mainly from the third quarter of the 6th to the first quarter of the 5th century BCE breaks new grounds in view of a better interpretation of the colony’s economic development during the later archaic period. Furthermore, it underlines Himera’s prominent position within the wider frame of regional economic interaction.

scholarly journals Dimensional characterization of heat flux in aquifers with horizontal water flow. Velocity estimation from temperature-depth profiles

2021 ◽  
Author(s):  
José Antonio Valera ◽  
Francisco Alhama
Keyword(s):  
Flow Velocity ◽  
Water Flow ◽  
Transfer Problem ◽  
Direct Determination ◽  
Velocity Estimation ◽  
Water Flow Velocity ◽  
Dimensionless Groups ◽  
Selection Of

From the dimensionless governing equations obtained through an adequate selection of dependent and independent dimensionless variables, and the use of spatial discrimination, the dimensionless groups that govern the solution patterns of the heat transfer problem in aquifers with horizontal flow velocity are determined. As a boundary condition on the surface, the cases of constant and harmonic temperature are studied. The emergence of a characteristic length that, in its dimensionless form, depends on the deduced groups, allows the direct determination of the water flow from experimental measurements of the temperature profiles in the classical form of the inverse problem.

Determination of Antioxidant Activity In Vitro from Hyptis rhomboidea Mart. et Gal. Ethanol Extracts

2014 ◽  
Vol 472 ◽  
pp. 824-828 ◽  
Author(s):  
Xi Feng Li ◽  
Lu Tang ◽  
Nan Nan Zheng ◽  
Shuo An ◽  
You Xu Wang ◽  
...  
Keyword(s):  
Antioxidant Activity ◽  
Antioxidant Activities ◽  
Positive Control ◽  
Fresh Market ◽  
Ethanol Extracts ◽  
Dpph Scavenging ◽  
Selection Of

Antioxidants are the chemical substances that reduce or prevent oxidation. The present study aimed to assess in vitro antioxidant activities of ethanol extracts Hyptis rhomboidea. DPPH, ABTS and FRAP assays were used for characterization of antioxidant activities of several extracts. Eventually, the promising extract was the ethyl acetate (EAc) extract, The analyzed extracts exhibited that antioxidant activities varied considerably as function of extracts. The highest DPPH• scavenging ability was found in Y6-8，which was divided from EAc by silica column, the lowest IC50 value (6.9μg/ml), followed by Y6-9 (11.7μg/ml) and the TEAC of Y6-8 and Y6-9were greater than positive control trolox. The same tendency was observed using ABTS and FRAP assays. These results suggest that the H. rhomboidea shows a better antioxidant activity, especially in the EAc extract and these results would be a guide in the selection of potential cultivars which are used in food and chemical industry and fresh market.

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