TA仪器+Affinity ITC+等温滴定微量热仪

Solution calorimetry measures the heat of solution of a solid dissolving into a solvent. Hogan & Buckton (2000) have shown there is a measurable difference in the enthalpies of solution between the amorphous and crystalline form of a material. Typically, the dissolution of a crystalline material is an endothermic process and exothermic for an amorphous material. Such an ability to differentiate between the amorphous and crystalline form of a material is important to formulators of solid dosage forms for drug delivery. In all of these forms the assessment of the order or possible disorder associated with the solid material is vital for stability and compatibility testing. Generally the presence of amorphous material influences the physical integrity and usually the kinetics of degradation. Often, only a small amount of amorphous material can produce severe effects on the stability of a particular formulation.

Materials such as coals, coal products (e.g., chars, briguettes) and activated carbons are a self-heating hazard in storage and transportation. Accordingly, work was carried out in the 1970’s (1,2) largely at the Fire Research Station in the UK, which led to the formulation of a test (3,4) for predicting whether or not a particular coal or carbon material was a self-heating hazard. The test wich is very widely used up to the present time, focuses on the quantity critical ignition temperature, and the present author has shown (5) that this is not the most suitable quantity around which to build such a test. The critical ignition temperature provides an indication of self-heating hazards only in the limit where all susceptible materials have the same activation energy (usual symbol E, units J mol-1) for their reaction with atmospheric oxygen. This is very far from the truth, and consequently the tests are unreliable.

通过活体组织提供一个直接的的定量检测同时测量热率产生和氧气利用率，意味着在代谢状态下面对改变生理和环境条件检测细微变化。The ratio of calorimetrically measured heat flux to the respirometrically measured oxygen flux is often called the “calorimetric-respirometric ratio” or CR ratio.

The expression or replication of genes is affected by the binding of small molecule ligands and proteins to nucleic acid sequences. Such binding events are critical for the physiological integrity of organisms and therefore are of fundamental interest to life scientists. Recently, the thermodynamics driving these interactions have also become important to pharmaceutical scientists investigating the anticancer, antibacterial and antiviral potential of nucleic acid/ligand interactions. In addition, as the number of diseases identified as being due to a malfunction of cellular control processes increases, the possibility of treating disorders by manipulating gene expression is further focusing attention on the thermodynamics underlying nucleic acid binding affinity and specificity.

Simultaneous measurements of the rates of heat production and oxygen utilization by living tissues provides a direct quantitative means of detecting subtle changes in metabolic state in the face of altered physiological and environmental conditions. The ratio of calorimetrically measured heat flux to the respirometrically measured oxygen flux is often called the “calorimetric-respirometric ratio” or CR ratio. This can be used to partition total metabolic energy flux into its aerobic and anaerobic components by comparison of the experimental CR ratio with the theoretical “oxycaloric equivalent” for fully aerobic respiration. Such information is most important when evaluating the physiological effect of environmental stress.

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.

When two proteins interact and bind, conformational changes in the proteins, and rearrangement of the solvent in the vicinity of the binding site, result in the absorption or generation of heat. Quantification of this reaction heat by ITC provides a complete thermodynamic description of the binding interaction, the stoichiometry of binding, and the association constant; in addition, if structural information is available, the contributions of specific amino acids mediating the binding event can be identified and their thermodynamic contributions quantified.

Continuous isothermal titration calorimetry (cITC) is ideal for studying rapid binding events typical of many biological recognition and binding reactions. The binding constant, stoichiometry and enthalpy of the reaction are accurately determined in significantly less time than that required using incremental isothermal titration calorimetry.