Measuring Enzymatic Stability by Isothermal Titration Calorimetry

Using enzymes as catalysts in reactions can reduce the amount of energy needed for the reaction, and reduce toxic byproducts. However, the use of enzymes in industry is limited, because enzymes are not stable and can be expensive. This protocol could reduce the amount of time that it takes to determine if conditions are deleterious to enzyme activity, or if modifications to the enzyme have increased its stability.

The main advantage of this technique is that it requires fewer man hours than traditional enzyme stability assays. Additionally, this protocol can be used with a wide range of enzymes because it can be used with any reaction that releases or absorbs heat. To begin, prepare 0.1 molar sodium acetate buffer.

Measure 800 milliliters of distilled water in a 1, 000 milliliter graduated beaker. Then, weigh 8.2 grams of anhydrous sodium acetate, and add it to the beaker. Place the beaker on a stir plate.

Place a stir rod into the beaker, and turn on the stir plate to stir it until completely dissolved. When the anhydrous sodium acetate is completely dissolved, use a calibrated pH meter to measure the pH of the solution. Use a pipette to add 1 molar hydrochloric acid or sodium hydroxide accordingly to obtain the desired pH at 4.6.

Add distilled water into the beaker until the total volume reaches 1, 000 milliliters. To prepare the enzyme solution, in a 15 milliliter graduated cylinder, measure 8 milliliters of the 0.1 molar sodium acetate buffer. Then, add the buffer solution into a 15 milliliter conical tube with enzyme, and shake vigorously until the enzyme has dissolved.

Add more buffer solution until the total volume reaches 10 milliliters. Store the enzyme solution at 4 degrees Celsius until use. To prepare the substrate solution, weigh out the substrate, and place it into a 100 milliliter glass beaker.

Use a graduated cylinder to measure 20 milliliters of the buffer solution, and then add it to the glass beaker. Place the beaker on a stir plate, and place a magnetic stir rod into the beaker. Turn on the heat, and adjust the stirring speed accordingly.

Allow stirring to continue until the substrate has dissolved. Pour the substrate solution into a 15 milliliter conical tube, and add the sodium acetate buffer previously prepared, until the total volume is 45 milliliters. Shake the tube to mix.

Store the substrate solution at room temperature until use. On the computer, open ITC Run and click setup. Click stirring rate, and set to 350 rpm.

Check the syringe size is at 50 microliters. Set the temperature to 55 degrees Celsius, and press update. Wait for at least one hour before proceeding, to allow enough time for the ITC instrument to heat up or cool down as needed.

Before loading the enzyme, ensure that the reference cell is loaded with 350 microliters of distilled water. To clean the sample cell, fill the loading syringe with 500 microliters of 2 percent cleaning solution. Carefully insert the needle into the sample cell, fill the cell, and slowly remove the liquid using the same syringe.

Dispose the liquid into a beaker. Repeat this step twice with 2 percent cleaning solution, three times with 70 percent ethanol, and then wash ten times with distilled water. After the sample cell has been cleaned, fill the loading syringe with 450 microliters of enzyme solution.

Carefully insert the needle all the way to the bottom of the sample cell, and slowly press the plunger down to the 100 microliter line to prevent formation of air bubbles. Then, wash the 50 microliter titration syringe with distilled water three times by placing the needle tip into the water to slowly take up the water into the syringe, then dispensing the water into a waste container. Rinse with the substrate solution three times to remove residual water.

After that, fill the titration syringe with substrate solution by drawing the solution up until the syringe is full without any air bubbles. With the syringe still in the substrate solution, remove the plunger, and allow approximately 2 microliters of air to enter the top of the syringe, and reinsert the plunger. Remove the burette handle of the ITC, place the syringe inside the burette handle, and screw until tight.

Wipe the tip of the titration syringe with a lint-free tissue, and then carefully place the burette handle into the ITC instrument, and lock it in place. For the experiment setup, select incremental titration. Click insert to set up the injections.

Adjust the injection interval to 5, 400 seconds, injection volume to 4 microliters, and number of injections to four. Press OK to confirm settings. In the equilibration box, select auto equilibrate and large expected heats.

Set the initial baseline to 300 seconds. To start the run, click the start symbol next to stirring rate, and then click start, which is located next to the wrench symbol. Save the file, and allow the instrument to run.

To analyze data, open the file in Nano Analyze. Click data, and select data columns. Select all data, copy, and then paste the data into Microsoft Excel.

Adjust 0 baseline by adding the value required at 300 seconds to make it 0. Apply this correction to the entire column of heat rate values. Then, plot graphs of the minimum or maximum values against the time at which the value occurred during the titration.

In this protocol, ITC data traces of lactase activity is shown with four sequential injections of lactose into a lactase solution in pH 4.6 buffer at 55 degrees Celsius, 45 degrees Celsius, 35 degrees Celsius, and 25 degrees Celsius. A no enzyme control was performed at 55 degrees Celsius for the heat of mixing. For each injection of enzyme, there is an initial exothermic heat of mixing, and subsequently, the lactase catalyzed endothermic hydrolysis of lactose occurs until the reaction is completed, and the heat rate returns to the baseline.

The endothermic peak minimum following each injection, is a little less than the preceding injection because of the decrease of enzymatic activity. As expected, the enzyme stability for each injection decreases with increasing temperature. The control experiment shows lactase activity at 25 degrees Celsius had an 8 percent decrease in enzyme activity, due to dilution after four injections.

Considering the fourth injection shows a 73 percent decrease in the assay, the actual loss of enzyme activity was therefore 65 percent. Thus, the dilution of the lactase resulted from each injection, had a relatively small effect of 11 percent on enzyme activity. It is important that the buffer used to make the enzyme and substrate solutions are matched as closely as possible.

Mismatch of pH or concentration can result in a larger heat of mixing which can mask the reaction heat. You can apply this protocol to study of enzymes and determine their stability by measuring the rate of heat released or absorbed during the course of a reaction.