A flow microcalorimetric method for enzyme activity measurements: Application to dihydrofolate reductase

Enzyme Kinetics Determined Using Calorimetry: A General Assay for Enzyme Activity?

“…Changes in thermal power (left panel) were converted to enzyme turnover (right panel) using Eq. [5] and an experimentally determined ⌬H app (Ϫ16 kcal/mol). Rate data for the cooperative hydrolysis of ATP were fit to the Hill equation (see Fig.…”

“…Continuous rate measurements following a single injection of substrate (typically at concentrations much greater than the K m ) can be made by monitoring the chemical thermal power as substrate is completely consumed. Figure 3 5 The time required between injections was kept to a minimum in order to maintain pseudo-first-order reaction conditions. In general, one only needs to collect thermal power measurements long enough to prevent the dilution heats from affecting the measurements.…”

“…1A); thus data collection at each substrate concentration was truncated after 3 min, and another injection was made. 5 Enzyme reaction rates were obtained from the data in Fig. 2A by measuring the difference between the original baseline and the new baseline after substrate addition.…”

“…These earlier studies, however, did not utilize current features of modern power-compensated titration calorimeters, including (i) the observable, thermal power is directly proportional to the rate (4,5) and need not be corrected for the time constant of the calorimeter, simplifying data analysis; (ii) multiple additions of substrate allow multiple rate determinations in a single experiment (under pseudo-first-order conditions); and (iii) low amounts of enzyme are required. We demonstrate two methods that exploit the high sensitivity and rapid response time of modern powercompensated isothermal titration calorimeters (6,7) and present kinetic analysis of the activity of biologically relevant enzymes from every EC classification.…”

Flow-microcalorimetric determination of enzymatic activities of the Trichoderma viridae-cellulase complex

“…Many enthalpimetric methods concerning the analysis of enzymatic reactions have been described during the last twenty years [2][3][4][5][6][7][8][9][10]. The use of calorimetry in the determination of enzymatic activities has many atlractive features, such as the possibility of continuous-flow operation, insensivity to the optical properties of the sample, simple procedures, a general detection principle, a dynamic monitoring range of several orders of magnitude in substrate concentrations, and high sensitivity.…”