Theoretical and Experimental Considerations for a Rapid and High Throughput Measurement of Catalase In Vitro

A rapid and high throughput protocol to measure the catalase activity in vitro has been designed. Catalase is an enzyme with unusual kinetic properties because it does not follow the standard Michaelis–Menten model and is inactivated by H2O2. This makes the analysis of the two rate equations of the second-ordered reactions of the kinetic model rather complex. A two-degree polynomial fitting of the experimental data is proposed after transforming the exponential form of the integrated rate equation of the [H2O2] into a polynomial using the Taylor series. The fitting is validated by establishing an experimental linear relationship between the initial rate of the H2O2 decomposition and the protein concentration, regardless of the suicide inactivation that catalase might undergo beyond t > 0. In addition, experimental considerations are taken into account to avoid statistical bias in the analysis of the catalase activity. ANOVA analyses show that the proposed protocol can be utilized to measure the initial rate of the H2O2 decomposition by catalase in 32 samples in triplicates if kept below 8 mM min−1 in the microplate wells. These kinetic and statistical analyses can pave the way for other antioxidant enzyme activity assays in microplate readers at small scale and low cost.

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A small scale in vitro system for high throughput gas production analysis – A comparison with the Hohenheim gas test

Animal Feed Science and Technology ◽

10.1016/j.anifeedsci.2018.04.001 ◽

2018 ◽

Vol 241 ◽

pp. 8-14 ◽

Cited By ~ 1

Author(s):

Karola Elberg ◽

Patrick Steuer ◽

Ute Habermann ◽

Jürgen Lenz ◽

Michael Nelles ◽

...

Keyword(s):

High Throughput ◽

Gas Production ◽

Small Scale ◽

In Vitro System ◽

Production Analysis

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Studies on the mechanism and kinetics of the 2-oxoglutarate dehydrogenase system from pig heart

Biochemical Journal ◽

10.1042/bj1610569 ◽

1977 ◽

Vol 161 (3) ◽

pp. 569-581 ◽

Cited By ~ 25

Author(s):

C L McMinn ◽

J H Ottaway

Keyword(s):

Initial Rate ◽

Optimization Technique ◽

Random Order ◽

The Other ◽

Rate Equations ◽

Kinetic Properties ◽

Ping Pong ◽

System 2 ◽

Dehydrogenase System ◽

Oxoglutarate Dehydrogenase

1. The kinetic properties of the 2-oxoglutarate dehydrogenase system were investigated. To this end, initial-velocity studies were carried out by the method of Fromm [(1967) Biochim. Biophys. Acta 139, 221-230]. Reciprocal plots of the results did not agree with those expected for the Hexa Uni Ping Pong mechanism previously proposed for the system. 2. The measured initial velocities were fitted to initial-rate equations corresponding to several possible mechanisms by using a computer optimization technique. Statistical analyses performed on the results of the optimization studies indicated that one mechanism was a significantly better fit to the experimental data than the other mechanisms tested. This mechanism is one in which there is a random order of binding of NAD+ and CoA and release of succinyl-CoA, although the binding of 2-oxoglutarate and release of CO2 is still given a Ping Pong mechanism, which precedes the binding of the other substrates. These conclusions were supported by NADH-inhibition studies. 3. The usefulness of the method of fitting initial-rate data to rate equations and the applicability of the proposed enzymic mechanism to the enzyme complex are discussed.

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Molecules incorporating a benzothiazole core scaffold inhibit the N-myristoyltransferase of Plasmodium falciparum

Biochemical Journal ◽

10.1042/bj20070692 ◽

2007 ◽

Vol 408 (2) ◽

pp. 173-180 ◽

Cited By ~ 45

Author(s):

Paul W. Bowyer ◽

Ruwani S. Gunaratne ◽

Munira Grainger ◽

Chrislaine Withers-Martinez ◽

Sasala R. Wickramsinghe ◽

...

Keyword(s):

Escherichia Coli ◽

Plasmodium Falciparum ◽

High Throughput ◽

High Throughput Screening ◽

Small Scale ◽

Expression And Purification ◽

Purification System ◽

Pure Protein ◽

Ic50 Values

Recombinant N-myristoyltransferase of Plasmodium falciparum (termed PfNMT) has been used in the development of a SPA (scintillation proximity assay) suitable for automation and high-throughput screening of inhibitors against this enzyme. The ability to use the SPA has been facilitated by development of an expression and purification system which yields considerably improved quantities of soluble active recombinant PfNMT compared with previous studies. Specifically, yields of pure protein have been increased from 12 μg·l−1 to >400 μg·l−1 by use of a synthetic gene with codon usage optimized for expression in an Escherichia coli host. Preliminary small-scale ‘piggyback’ inhibitor studies using the SPA have identified a family of related molecules containing a core benzothiazole scaffold with IC50 values <50 μM, which demonstrate selectivity over human NMT1. Two of these compounds, when tested against cultured parasites in vitro, reduced parasitaemia by >80% at a concentration of 10 μM.

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Engineered illumination devices for optogenetic control of cellular signaling dynamics

10.1101/675892 ◽

2019 ◽

Cited By ~ 1

Author(s):

Nicole A. Repina ◽

Thomas McClave ◽

Xiaoping Bao ◽

Ravi S. Kane ◽

David V. Schaffer

Keyword(s):

Cell Fate ◽

High Throughput ◽

Protein Interactions ◽

Cell Biology ◽

Low Cost ◽

Optical Design ◽

Embryonic Stem ◽

Protein Protein Interactions ◽

Optogenetic Control

ABSTRACTSpatially and temporally varying patterns of morphogen signals during development drive cell fate specification at the proper location and time. However, currentin vitromethods typically do not allow for precise, dynamic, spatiotemporal control of morphogen signaling and are thus insufficient to readily study how morphogen dynamics impact cell behavior. Here we show that optogenetic Wnt/β-catenin pathway activation can be controlled at user-defined intensities, temporal sequences, and spatial patterns using novel engineered illumination devices for optogenetic photostimulation and light activation at variable amplitudes (LAVA). The optical design of LAVA devices was optimized for uniform illumination of multi-well cell culture plates to enable high-throughput, spatiotemporal optogenetic activation of signaling pathways and protein-protein interactions. Using the LAVA devices, variation in light intensity induced a dose-dependent response in optoWnt activation and downstream Brachyury expression in human embryonic stem cells (hESCs). Furthermore, time-varying and spatially localized patterns of light revealed tissue patterning that models embryonic presentation of Wnt signalsin vitro. The engineered LAVA devices thus provide a low-cost, user-friendly method for high-throughput and spatiotemporal optogenetic control of cell signaling for applications in developmental and cell biology.

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Toward high-throughput predictive modeling of protein binding/unbinding kinetics

10.1101/024513 ◽

2015 ◽

Cited By ~ 3

Author(s):

See Hong Chiu ◽

Lei Xie

Keyword(s):

High Throughput ◽

Critical Role ◽

Conformational Dynamics ◽

Drug Efficacy ◽

Mode Analysis ◽

Computational Techniques ◽

Kinetic Properties ◽

Task Learning

One of the unaddressed challenges in drug discovery is that drug potency determined in vitro is not a reliable indicator of drug efficacy and toxicity in humans. Accumulated evidences suggest that the in vivo activity is more strongly correlated with the binding/unbinding kinetics than the equilibrium thermodynamics of protein-ligand interactions (PLI) in many cases. However, existing experimental and computational techniques are both insufficient in studying the molecular details of kinetics process of PLI. Consequently, we not only have limited mechanistic understanding of the kinetic process but also lack a practical platform for the high-throughput screening and optimization of drug leads based on their kinetic properties. Here we address this unmet need by integrating energetic and conformational dynamic features derived from molecular modeling with multi-task learning. To test our method, HIV-1 protease drug complexes are used as a model system. Our integrated model provides us with new insights into the molecular determinants of the kinetics of PLI. We find that the coherent coupling of conformational dynamics between protein and ligand may play a critical role in determining the kinetic rate constants of PLI. Furthermore, we demonstrated that Normal Mode Analysis (NMA) is an efficient method to capture conformational dynamics of the binding/unbinding kinetics. Coupled with the multi-task learning, we can predict combined kon and koff accurately with an accuracy of 74.35%. Thus, it is possible to screen and optimize compounds based on their kinetic property. Further development of such computational tools will bridge one of the critical missing links between in vitro drug screening and in vivo drug efficacy and toxicity.

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