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Calorimetric Enzymes Assays

With advanced technologies and extensive knowledge of enzymes, Creative Enzymes is engaged in providing reliable enzyme activity assays for both conventional and unique research needs. Our testing services are famous for the high quality and rapid turnaround, which are endorsed by a long list of experts and scientists. To facilitate various enzymatic research, Creative Enzymes offers a variety of approaches for enzyme activity measurement to support different research demands. Besides the relatively well-understood methods such as spectrophotometric and colorimetric assays, Creative Enzymes has introduced latest technology and cutting edge instruments to develop more advanced measurement. As one of the most accurate test methods, calorimetric assay has been utilized in several biological areas. We proudly launch the enzyme activity assay service using isothermal titration calorimetry.

Isothermal titration calorimeter (ITC) has been employed more widely compared to its early introduction into the industry two decades ago. Currently, ITC is a well adopted method to determine binding affinities to enzymes. The basic principle is the direct measurement of the enthalpy change for a bimolecular binding interaction at a constant temperature. This technique measures the heat released or absorbed over time, and is therefore a universal and convenient methodology to quantify the amount of reacting molecules such as the binding thermodynamics, as well as to measure the reaction rate.

The universal nature of the technique is reflected in the vast array of systems that have been interrogated using ITC to date. Recent reviews have described the utility of the technique in the study of protein–drug, drug–DNA, protein–DNA and protein–carbohydrate interactions. In addition, ITC is not restricted in any way by the molecular weight of the ligand. Compared to other activity analysis method, ITC has its advantages. Typically, the commonly used spectrophotometric and fluorescence assays work through measuring the light absorption or fluorescence of substrate or product to determine the enzyme activity. However, many substrates and products are not spectroscopically active. As for coupled assays, which works through coupling another reaction and uses the product of former reaction to be the substrate of the latter’s, reducing the accuracy of the determination because of the introduction of additional variables. In the research of enzyme inhibition by small ligands, traditional methods may alter the reliability since the absorption or fluorescence of small ligand itself. Stopped flow techniques are precise but time consuming and expensive for routine analysis. Applying the intrinsic property of almost all chemicals that the heat released or absorbed, ITC does not require any modification or labeling of the system under analysis. Moreover, this technique is straightforward and fast, requires small amount of material and can be performed in solution. ITC is a reliable and fast method to characterize enzymatic reactions, without the requirement of system modification or labeling.

However, there is no exception that every testing method has limitations. A good signal-to-noise ratio is obtained only if the reaction proceeds at a rate that allows the system to produce enough heat in the ITC cell to overcome the instrumental detection limit. This is usually achieved for systems with kcat higher than 1min-1. Given the intrinsic difference of every enzymatic system and the different heat produced by different reactions, the optimal running conditions cannot be quantified in advance. During the process, care must be applied to choose the experimental conditions according to the system under analysis. The extensive experiences in Creative Enzymes will contribute to establish proper analysis process in ITC assays. In addition, considering the high dependency of the equipment, Creative Enzymes only utilizes the first-in-class instruments to provide accurate results.

Schematic representation of isothermal titration calorimeter to study enzymatic reactions. Figure: Schematic representation of isothermal titration calorimeter to study enzymatic reactions.
Reference: Luca Mazzei et al. J. Vis. Exp. 2014, (86), e51487.

Leading improvement in enzyme activity assays, Creative Enzymes always takes it as the ultimate goal to provide more reliable tests for each customer. The results are guaranteed with superb quality and high reproducibility. Our ITC enzyme assay services will greatly expedite your research. In case you have any technical concern, please contact us for professional support.

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