Isothermal titration calorimetry (ITC) was used to measure the activity of the enzyme sarco/endoplasmic reticulum calcium APTase. ITC is a rapid and versatile method for the direct measurement of enzyme activity. In comparison to other methods, it does not dependent on coupled reactions and auxiliary enzymes since it detects enzyme activity directly by measuring the enthalpy of the reaction.  Another advantage of ITC is that it does not suffer from limitations due to the presence of light-absorbing inhibitors that could be problematic in spectroscopic assays.  Here, ITC experiments were carried out by titrating adenosine triphosphate (ATP) into a solution of SERCA and measuring the heat signals caused by the hydrolysis of ATP. Kinetic analysis provided the classic parameters Vmax and KM. Titrations were also performed in the presence of inhibitors, which allowed the determination of inhibition constants.

Application of isothermal titration calorimetry for the measurement of enzyme kinetics: The sarco/endoplasmic reticulum calcium ATPase

246th National Meeting (2013)

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