Real-Time Analysis of Enzyme Kinetics via Micro Parallel Liquid Chromatography

Abstract

A generic method for real-time monitoring of enzyme kinetics is described in this paper. This approach enables rapid development of assays for high-throughput screening or reaction monitoring in the linear range of the enzyme kinetic curve. In this paper, we used protein kinase A and kemptide (a well-studied assay system) to demonstrate assay optimization by using micro parallel liquid chromatography. The optimal substrate and enzyme concentrations were determined rapidly and conveniently compared with the traditional methods for determining these parameters. Additionally, the data collected from the same experiment permitted calculations of K (m) for the substrate, V (max), and time-course study. In general, this approach provides two advantages. First, the broad ranges of detectable product conversions facilitate selection and implementation of assay conditions for high-throughput screening. Second, the system permits determination of 50% inhibitory concentration values at less than 1% conversion of substrate to product, thereby validating screening hits in the linear range of the enzyme kinetic curve. Overall, this optimization process can be done in less than 8 h. To demonstrate the ability to monitor a wide range of assay conditions, we varied initial concentrations over eight orders of magnitude within a single experiment. Compared with a classical enzyme kinetics study, this method significantly speeds the target validation process and reduces time associated with assay development and high-throughput screening implementation.

Spectroscopic studies and antibacterial activities of pure and various levels of Cu-doped BaSO4 nanoparticles

“…Barium sulphate, which is commonly known as Barite and inert in lots chemical reactions, has been extensively used in many areas such as catalyst carriers [17,18], adsorbent materials [19,20], printing ink [21], engineering plastics [22], biomedical devices [23], fillers and add-ons in polymers [22,24]. BaSO 4 is used in paints and coatings for stabilization of pigments against flocculation and show high transparency.…”

Mapping phospho-catalytic dependencies of therapy-resistant tumours reveals actionable vulnerabilities

“…Mass spectrometry techniques [10][11][12][13][14][15][16][17] , somtimes combined with kinase inhibitors [18][19][20][21] , allow the detection of raw amounts of (phospho-)proteins, but remain restricted due to cost, equipment and protocols. Alternatively, generic amino acid sequences are used as individual biochemical probes to directly detect kinases' phospho-catalytic activity in radioactive labelling assays, microfluidic electrophoresis systems, adenosine triphosphate (ATP) consumption tests, hybrid peptide/phospho-antibody platforms, or surface plasmon resonance (SPR) and fluorescence resonance energy transfer (FRET) techniques [22][23][24][25][26][27][28][29] . However, readouts from these approaches rely on broad-spectrum consensus peptides originally designed for oneprobe-to-many-kinases detection methods, which are well suited for pharmacological drug screens, but not intended to specifically identify or differentiate between kinases' activity in biological extracts.…”

Microscale separation methods for enzyme kinetics assays

“…The crude extract was first separated by CLC. The eluent was mixed on-line with AchE and acetylcholine and products detected by ESI-MS. A special arrangement of CLC, micro parallel liquid chromatography, was used by Wu et al [80,81] for studying protein kinase A. Analyses take place parallel in 24 separation capillaries, which makes the method rapid with high throughput.…”