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.

A novel microfluidic device was designed and developed to miniaturize, multiplex, and automate serial dilution and three-reagent dose-response assays using submicroliter quantities of reagents. This prototype microfluidic device can be used to measure enzyme kinetics and to test a chemical lead's response to a target by fluorescent readout using common plate readers and detection systems. The prototype microfluidic system yielded serial dilution and dose-response assay data comparable to results obtained from manual titrations and reagent additions performed using a microwell plate. Enzyme kinetics were highly reproducible using these devices, although Michaelis-Menten kinetics results differed from those obtained in the microwell plate. In all cases reported here, assays performed on the microfluidic format required lower volumes of reagents compared with the microwell plate. In addition to savings in reagent consumption, the microfluidic devices and bench-top instruments offer additional advantages over conventional solutions, including a small footprint and compatibility with commercially available fluorescence detectors. Future directions for the prototype technology are discussed.

The Nanostream (Pasadena, CA) Veloce system, together with 24-column Brio cartridges, offers a novel approach to micro parallel liquid chromatography (μPLC). This system allows users to achieve unprecedented throughput for standard assays while matching the performance of conventional LC instrumentation, thus enabling routine compound purity assessment and physiochemical property profiling early in the drug discovery and development process. The Veloce system—which includes instrumentation, software, and replaceable microfluidic cartridges—incorporates pressure-driven flow to achieve chromatograms comparable to conventional high performance liquid chromatography (HPLC) instrumentation for a broad class of analytical applications while offering a dramatic increase in sample analysis capacity. The system enables parallel chromatographic separations and simultaneous, real-time UV detection. Each Nanostream Brio cartridge, made of polymeric materials, incorporates 24 columns packed with standard (C-18) stationary phase material to achieve reverse phase separations. Mixing and distribution of the mobile phase to each of the 24 columns is precisely controlled in each cartridge. The system provides an ideal platform to accelerate assessment of compound purity and physicochemical properties (i.e., log P, CHI, etc.) for a large number of compounds. In addition, the 24-fold increase in sample analysis capacity allows standard curve generation and simultaneous analysis of multiple replicates of samples in a single run. (JALA 2004;9:185-91)

Phosphorylation is central to signal transduction in living organisms. The specificity of phosphorylation ensures signaling fidelity. Understanding substrate specificity is essential for novel assay development in drug discovery. In this study, we have developed an innovative approach to study protein kinase and its substrate specificity. Using 24 micro parallel liquid chromatography, we studied the reaction kinetics for two different peptide substrates commonly associated with protein kinase A (PKA): Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Glu) and CREBtide (Lys-Arg-Arg-Glu-Ile-Leu-Ser-Arg-Arg-Pro-Ser-Tyr-Arg). The phosphorylation of each substrate was monitored in real time, and the kinetic parameters (V(max), K(m), k(cat), and k(cat) K(m)) were determined for a variety of initial conditions. The results from several kinetic experiments indicated that Kemptide had higher V(max) and k(cat) values compared to CREBtide under the same assay conditions. However, both substrates had a similar k cat)/K(m) value, suggesting that both substrates have similar specificity constants for PKA. We further analyzed the reaction kinetics of ATP for both PKA/substrate complexes. Interestingly, we found that there was a fivefold difference in the specificity constants for ATP affinity to the two complexes, suggesting that even though the sequence differences between the two substrates do not affect their independent interactions with PKA, the differences do have a secondary effect on each enzyme's interaction with ATP and significantly alter the ATP consumption and thus phosphorylation. This novel approach has a broad application for studying enzyme functions and enzyme/substrate specificity.