Abstract
Recent developments in microfluidic and nanofluidic technologies have resulted in development of new chip-based microfluidic calorimeters with potential use in different fields. One application would be the accurate high-throughput measurement of enzyme activity. Calorimetry is a generic way to measure activity of enzymes, but unlike conventional calorimeters, chip-based calorimeters can be easily automated and implemented in high-throughput screening platforms. However, application of chip-based microfluidic calorimeters to measure enzyme activity has been limited due to problems associated with miniaturization such as incomplete mixing and a decrease in volumetric heat generated. To address these problems we introduced a calibration method and devised a convenient protocol for using a chip-based microfluidic calorimeter. Using the new calibration method, the progress curve of alkaline phosphatase, which has product inhibition for phosphate, measured by the calorimeter was the same as that recorded by UV-visible spectroscopy. Our results may enable use of current chip-based microfluidic calorimeters in a simple manner as a tool for high-throughput screening of enzyme activity with potential applications in drug discovery and enzyme engineering.
Highlights
High-throughput measurement of enzyme activity is dependent on assays that enable time-resolved data collection to record activity of enzymes for a few seconds after addition of substrate
We showed that the activity of alkaline phosphatase with para-nitrophenyl phosphate (PNPP) as measured by microfluidic calorimeter matched well to that measured by UV-visible spectrometry
We predict that our method to calibrate the microfluidic calorimeter and our suggested experimental design will allow convenient use of microfluidic calorimeters for high-throughput and accurate measurement of enzyme activity using natural substrates for screening purposes, e.g. for screening of enzyme inhibitors as possible drug candidates or in identifying new mutants of an enzyme with improved activity
Summary
High-throughput measurement of enzyme activity is dependent on assays that enable time-resolved data collection to record activity of enzymes for a few seconds after addition of substrate. A method for timeresolved initial rate measurements using isothermal titration calorimetry (ITC) has been developed as a generic assay for the activity of enzymes This method is based on a simple calibration reaction and enables accurate initial rate measurements by only recording a few early data points for the heat generated by an enzymatic reaction [8]. This method enables fundamental studies of many enzymes with their natural substrates and provides a tool for applying ITC instruments with high-throughput capability in measuring enzyme activity, still enzyme activity measurements using modern ITC instruments are significantly slower compared to those using UV-visible or fluorescence spectroscopy. Practical application of this calibration method was demonstrated for alkaline phosphatase, which catalyses the dephosphorylation of different substrates [16] and is a target for drug discovery [17]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
