蛋白质中活性检测方案(差示扫描量热)

Microcalorimetric instruments and methods are now essential tools for the general understanding of binding thermodynamics of biological macromolecules and specific biological systems. Provided that the enthalpy of binding is of appropriate magnitude, it is possible to determine affinities, KD, in the order of 10 nM (K=l08 M-1) for 1:1 complexes. One of the most important issues with ITC is the time needed to obtain a full binding isotherm. ITC inherently suffers from the relatively small number of binding experiments that are possible to perform per day. The focus has so far been on improving calorimetric response time of the instruments by various means, like assigning instrumental time constants and applying Tian’s equation to dynamically correct the raw calorimetric signal from a fast step-wise titration. Notably, nothing has so far been done to improve the experimental procedure. At the moment 2-3 h is needed to complete an ITC binding experiment. The number of data points that can be obtained, 20-30 points, limits the range of equilibrium constants that can be resolved from the data. For ITC this range is 1 ≤ KCM ≤ 1000, where K is the equilibrium constant and CM is the concentration of the reactant in the vessel. In this Application Note we show the possibility to shorten the time of an ITC experiment by slow continuous titration into the calorimetric vessel, cITC.