Measuring Enzymatic Stability by Isothermal Titration Calorimetry

Summary

The thermal stability of enzyme activity is readily measured by isothermal titration calorimetry (ITC). Most protein stability assays currently used measure protein unfolding, but do not provide information about enzymatic activity. ITC enables direct determination of the effect of enzyme modifications on the stability of enzyme activity.

Abstract

This work demonstrates a new method for measuring the stability of enzyme activity by isothermal titration calorimetry (ITC). The peak heat rate observed after a single injection of the substrate solution into an enzyme solution is correlated with enzyme activity. Multiple injections of the substrate into the same enzyme solution over time show the loss of enzyme activity. The assay is autonomous, requiring very little personnel time, and is applicable to most media and enzymes.   

Introduction

Enzymes are proteins capable of catalyzing a wide array of organic reactions. Most enzymes function in aqueous solution at near neutral pH thus avoiding the use of harsh solvents. Because of their high selectivity, enzyme catalyzed reactions produce fewer (in some cases no byproducts) byproducts than non-selective catalysts such as acids and bases¹. This is especially relevant in food manufacturing where all chemical reactions must be done so the final product is safe for human consumption. Currently, enzymes are used to produce high fructose corn syrup², cheese³, beer⁴, lactose-free milk⁵, and other important food products. While this paper focuses on enzyme use in the food industry, there are many other uses for enzymes including in green chemistry and drug synthesis. 

The utility of enzymes is limited by the stability of enzyme activity, which depends on maintaining the three-dimensional structure of the enzyme. The enzyme structure can be stabilized by modifications such as PEGylation⁶, immobilization on a solid support⁷, genetic modifications⁸, and formulations. Currently, enzyme stability is typically measured by differential scanning calorimetry (DSC) and endpoint enzyme activity assays⁹. DSC measures the temperature at which an enzyme unfolds; the higher the temperature, the more stable the structure. However, loss of activity often occurs at a lower temperature than required to unfold the enzyme or domains within the enzyme¹⁰. Therefore, DSC is not sufficient to determine whether an enzyme modification increases the stability of enzyme activity. Endpoint enzyme assays are usually time intensive, require multiple samples, and often involve a coupled colorimetric reaction that is not applicable to highly colored or opaque solutions or suspensions.

This work demonstrates a method for direct measurement of the stability of enzyme activity by isothermal titration calorimetry (ITC). ITC measures the rate of heat released or absorbed during the course of a reaction. Since nearly all reactions produce or absorb heat, ITC can be used for most enzyme-catalyzed reactions, including reactions that do not have a coupled reaction or occur in opaque media such as milk. ITC has been used for many decades to measure chemical kinetic parameters for many kinds of reactions, but the protocol presented here focuses on using ITC to measure the peak heat rate of enzyme-catalyzed reactions and demonstrates that enzyme activity is linearly correlated with the peak heat rate. ITC measurements of peak heat rates are mostly autonomous and require very little personnel time to setup and analyze. 

Protocol

1. Preparing samples

1. 1,000 mL of 0.1 M sodium acetate buffer at pH 4.6
   1. Measure 800 mL of distilled water in a 1,000 mL graduated beaker.
   2. Weigh 8.2 g of anhydrous sodium acetate and add it to the beaker.
   3. Place the beaker on a stir plate, place a stir rod into the beaker, turn on the stir plate and stir until completely dissolved.
   4. When the anhydrous sodium acetate is completely dissolved, measure the pH of the solution with a calibrated pH meter.
   5. Add 1 M HCl or NaOH accordingly to obtain the desired pH 4.6.
   6. Add distilled water until the total volume is 1,000 mL.
   7. Store at room temperature until use.
2. Enzyme solution
   1. Prepare 10 mL of the enzyme solution within the 10-30 mg/mL range by first measuring 8 mL of the 0.1 M sodium acetate buffer pH 4.6 in a 15 mL graduated cylinder. 
   2. Add the buffer solution into a 15 mL conical tube with enzyme and shake vigorously until the enzyme has dissolved.
   3. Add more buffer solution until the total volume is 10 mL.
   4. Store the enzyme solution at 4 °C until use.
3. Substrate solution
   1. To prepare a substrate solution within the 300-600 mM range, calculate the amount of substrate needed in grams to make the desired concentration.
   2. Weigh out the substrate and place into a 100 mL glass beaker
   3. Measure 20 mL of the buffer solution using a 25 mL graduated cylinder and then add it to the glass beaker.
   4. 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.
   5. Allow stirring to continue until the substrate has dissolved.
   6. Pour the substrate solution into a 50 mL conical tube and add 0.1 M sodium acetate buffer pH 4.6 until the total volume is 45 mL. Mix by shaking.
   7. Store the substrate solution at room temperature until use.

2. Performing the experiment

1. Preparing the ITC instrument 
   1. Ensure that the reference cell is loaded with 350 µL of distilled water. Before loading the enzyme into the sample cell, verify that the sample cell has been cleaned.
   2. Cleaning protocol- Fill the loading syringe with 500 µL of 2% cleaning solution (Table of Materials), 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% cleaning solution (Table of Materials), three times with 70% ethanol and then wash ten times with distilled water.
   3. Fill the loading syringe with 450 µL of enzyme solution, carefully insert the needle all the way to the bottom of the sample cell and press the plunger down to the 100 µL line slowly to prevent formation of air bubbles.
   4. Wash the 50 µL titration syringe with distilled water three times by placing the needle tip into water then slowly taking up the water into the syringe, then dispensing the water into a waste container.
   5. Remove residual water by rinsing with substrate solution three times.
   6. Fill the titration syringe with substrate solution by drawing the solution up until the syringe is full without any air bubbles. 
   7. With the syringe still in the substrate solution, remove the plunger and allow approximately 2 µL of air to enter the top of the syringe and reinsert the plunger.
   8. Remove the buret handle of the ITC, place the syringe inside the buret handle and screw until tight. 
   9. Wipe the tip of the stirrer with a lint-free tissue, then carefully place the buret handle into the ITC instrument and lock it in place.

3. Setting up ITCrun 

1. On the computer, open ITCrun and click Set up.
2. Click Stirring rate and set to 350 RPM. Check the syringe size (µL) and ensure it is at 50 µL.
3. Set the temperature and press Update. It is recommended that this step be performed at least 1 h before preparing the ITC. This allows enough time for the instrument to heat up or cool down as needed.
4. For the experiment setup, select incremental titration.
5. Click Insert to setup the injections. Adjust the injection interval to 5,400 s, injection volume (µL) to 4 and number of injections to 4. Press OK to confirm settings.
6. In the Equilibration box, select Auto-equilibrate and Large expected heats. (If the expected heats are small, one can select Small under expected heats; however, this will increase the equilibration time.)
7. Set the initial baseline to 300 s.
8. To start the run, click the Start symbol next to Stirring rate and then click Start which is located next to the wrench symbol.
9. Save the file and allow the instrument to run.

4. Analyzing data

1. Open the file in NanoAnalyze. Click Data and select Data columns.
2. Select all data, copy and then paste the data into Microsoft Excel.
3. Adjust zero baseline by adding the value required at 300 s to make it zero. Apply this correction to the entire column of heat rate values. 
4. Find the minimum or maximum value of the heat rate for each injection by using the equation: =MIN(cell:cell) or MAX(cell:cell). Each data point represents the peak enzymatic activity of the enzyme at each injection.
5. Plot graphs of the MIN or MAX values against the time at which the value occurred during the titration.

Results

The representative results in Figure 1 and Figure 5 show data from two enzymes, lactase and invertase. Lactase and invertase catalyze the hydrolysis of a disaccharide into two monosaccharides, endothermically and exothermically, respectively. Both enzymatic reactions were run at concentrations that precluded saturation of the enzyme. 

The lactase data demonstrate how ITC data can be used to estimate enzyme stability. Four sequential 4...

Discussion

A major advantage of the ITC enzyme stability assay described here is automation. Once all the appropriate buffers and solutions are made, the set-up time for each assay is approximately 15 min for the person doing the assay. In contrast, the conventional assays for invertase and lactase activity require about 2 h with continual involvement of the person doing the assay and many enzymatic activity assays take considerably more person-hours. In a previous publication, we have demonstrated how data from the ITC method comp...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
a-Lactose	Fisher Scientific	unknown (too old)	500g
Sodium Acetate, Anhydrous 99% min	Alfa Aesar	A13184-30	250g
Lactase	MP Bio	100780	5g
Hydrocholric Acid Solution, 1N	Fisher Scientific	SA48-500	500mL
Benchtop Meter- pH	VWR	89231-622
Ethanol 70%	Fisher Scientific	BP8231GAL	1gallon
Micro-90	Fisher Scientific	NC024628	1L (cleaning solution)