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, la....

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. St....

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)