The following experiment describes a reliable, fast, and label free method that employs isothermal titration calorimetry to quantitatively determine the thermodynamics and kinetics of enzymatic reactions in solution using heat released and absorbed over time as an internal probe. This is achieved by allowing the enzymatic reaction to occur in the instrument sample cell at a constant temperature and monitoring the deviation of the heat trace from the initial baseline. In a first experiment called Method one or M1, the substrate is injected into the enzyme solution and the heat for complete substrate conversion is measured allowing determination of the total molar enthalpy of the reaction.
In a second experiment called method two or M two, multiple injections of the substrate are performed, allowing measurement of the rate of heat production over time at different substrate concentrations. The results derived from the two calor metric experiments enabled determination of the kinetic parameters, kcat and km, assuming a meis menton enzymatic catalysis. The main advantage of using isothermal attrition calorimetry over other methods such as petro photometric assays that monitor product formation over time is that ITC does not require any modification and labeling of the system under analysis and needs very little amount of material as it uses the intrinsic heat of reaction as an internal probe demonstrating the procedure will be lusai.
A PhD student from our laboratory First dilute a concentrated solution of the enzyme with two milliliters of buffer to give a final enzyme concentration in the nano molar range for the M1 experiment. Then add 0.5 milliliters of buffer to a concentrated solution of the substrate to give a substrate concentration of at least three orders of magnitude higher than the enzyme concentration and above the km. For the M two experiment, prepare a two milliliter enzyme solution with a final concentration in the picaMolar to nanomolar range.
Dilute the substrate with 0.5 milliliters of buffer to give a substrate concentration in the millimolar range. After verifying that the sample cell and the injection syringe are cleaned according to the manufacturer's instructions, fill the loading syringe with distilled water. Then gently insert the needle in the sample cell, fill the cell and remove the liquid using the previously described method, wash the sample cell twice with distilled water and twice with buffer.
Slowly inject the enzyme solution into the cell until it spills out the top of the cell stem. To remove air bubbles trapped in the sample cell, produce a spurt of about 0.25 milliliters of solution into the cell. After producing the spurt two more times.
Place the needle of the loading syringe on the ledge between the cell stem and the cell port, and remove any excess solution. Start the VP viewer program and equilibrate the ITC instrument to a temperature three degrees Celsius below the desired experimental temperature. After filling the reference cell with distilled water in the same manner as before, link a plastic syringe to the fill port of the injection syringe using a thin silicon tube, place the injection syringe tip into water and fill the syringe until the water comes out of the top filling port indicating that the injection syringe is full.
Then remove the syringe tip from the water and draw up air to empty the injection syringe. Using the same procedure, wash the injection syringe with buffer and draw air through the system. Next, lace the substrate solution in a clean, narrow tube.
Position it under the syringe and insert the needle of the injection syringe in it completely. Fill the injection syringe, leaving a small amount of solution at the bottom of the tube from the computer interface. Press close fill port.
Once the silicon loading tube has been removed, press purge and refill to dislodge any air bubbles from the injection syringe and to expel them back into the bulk solution. Following this, wipe the sides of the injection syringe with a paper towel to remove any residual solution and place it in the sample cell. In the M1 experiment, set at least two additions of five to 30 microliters of substrate to verify the reproducibility.
Then set the spacing time between each injection large enough to ensure that the heat signal returns to the baseline before the next addition. In the M two experiment, set multiple injections of five to 10 microliters. Set the interval between the injections, allowing the system to stabilize the thermal power to the new baseline after each injection.
At this point, set the reference power to 20. Then define the experimental enzyme and substrate concentrations and choose a name for the experiment. Set temperature to 25 degrees Celsius and start the experiment.
Once the experiment is finished, clean the sample cell and the syringe according to the manufacturer's instructions. After opening the origin 7.0 software, click on the open button and select the file corresponding to the first injection in the heat trace of the M1 experiment. To determine the delta H of the reaction, integrate the first peak resulting from baseline deviation in the thermogram of the single injection.
M1 experiment. Then divide the obtained area value corresponding to the total heat of reaction expressed in micro calories by the final substrate, concentration in the sample cell expressed in micromolar and the sample cell volume expressed in liter. Repeat the same procedure for the second peak of the M1 experiment and average the values obtained for the two delta H measurements subsequently determine DQ over DT from the M two experiment by measuring the difference between the original baseline and the new baseline following each injection.
This can be done by converting the experimental data to reaction rates according to the following equation. Using the enthalpy value obtained in the M1 experiment. Then fit the data to the michalis menon equation in particular from the analysis program.
Click on the read data button and navigate to the folder where the M two experiment has been located. Then click on the scroll down arrow of the files of type and choose enzyme assay. Subsequently, click and open the file.
Do ITC of the M two experiment. After the enzyme assay dialogue box opens, select method two substrate only from the window in the delta H window indicate the value of delta H obtained in the M1 experiment. Next, click the zero y axis button and double click just before the first injection point.
Click the calculate rate button to plot the rate versus the substrate concentration. Finally, use the fit to model function to fit the curve and to obtain the kinetic constance. The lytic activity of cannavale and CMIS or JAK bean URIs was chosen as a representative reaction to demonstrate the applicability of ITC in quantitatively determining enzymatic catalysis.
A delta H value of negative 10.5 kilo calories per mole was measured in the M1 experiment By integrating the thermal power arising from the urea substrate injection into the urease solution and allowing the reaction to proceed to completion. The thermal power registered in the M two experiment was converted to the reaction rate using the following equation. Fit of the obtained data to the McKayla Cementin equation provided the kinetic parameters for JAK bean urease in 20 millimolar heaps.
pH seven as kcat equals 30, 200 per second, and KM equals 3.45 millimolar in agreement with previously reported data. The total heat change measured in the M1 experiment represents the sum of all events occurring during the reaction under analysis, and depends on the molar enthalpy of all the processes involved, and not only on the substrate to product conversion. In a generic process in which the reacting system acquires protons in a buffered solution, the buffer releases protons producing additional heat within the reaction cell.
Therefore, the measured delta H is apparent and includes the intrinsic delta H of the reaction, as well as the ionization enthalpy of the buffer and the number of exchanging protons according to the highlighted equation. Using this equation in M1 experiments, the number of exchanging protons and the intrinsic delta H can be determined by performing the same experiment in buffers with different ionization enthalpy. The overall urea hydrolysis reaction results in the acquisition of a proton from the buffer.
Accordingly, as described above, the heat of the reaction includes the contribution of buffer d protonation. In order to calculate the intrinsic delta age of the hydrolysis reaction, three M1 experiments were performed in different buffers, characterized by three known different enthalpy of ionization. The intrinsic enthalpy and the number of protons released from the buffer can be calculated from the intercept and the slope of the linear fit of the experimental data.
They were negative 14.7 kilo calories per mole and 0.98 respectively in full agreement with data previously reported. Applications of this procedure extends from basic to applied research and drug screening, as once the kinetic of enzymatic reaction have been determined. The experiment can be repeated in the presence of different kinds and concentrations of enzyme inhibitors in order to monitor inhibition, constants.
