Hoff Determination of Thermodynamic Parameters

The varit Hoff method has been the most commonly applied technique to determine thermodynamic parameters. A MEDLINE search of varit Hoff reveals over 500 publications between 1966 and 2002. The application to enzyme reaction is well known. More recently, this method has been applied to ligand-receptor inter- 

5) Bovine pancreatic RNAse-A, 2 -CMP free acid (98% purity), Na, K, Ca, Mg acetate, and glacial acetic acid (ACS or molecular biology grade) were purchased from commercial sources. The RNAse v as dissolved in deionized v ater and was dialyzed twice for 4 h (in 20 mL solution) in a stirred 1-L beaker maintained at 1.5 °C by immersion in an ice-bath. RNAse and salt stock solutions (in deionized water) were mixed such that the final concentrations were KCl (3 mM), CaCh (0.1 inM), NaAcetate (10 mM), K2PO4 (3 mM), MgSO4 (0.4 mM), and KAcetate (50 mM) adjusted to pH 5.5 by dropwise addition of 50 mM HAce-tate. The concentration of RNAse (0.04-0.05 mM), selected to be not much higher than the JQ of interaction with 2 -CMP, was-determined by quantitative UV spectrophotometry (277.5 nm extinction coefficient 

Some of the more likely encountered possible hmitations in thermodynamic analysis, particularly for ligand-receptor interactions, include fhe following  

Although thermodynamic parameters can be obtained for interaction mechanisms that are complex, interpretation of the results is greatly simplified when the interaction mechanism is simple. For example, tissues in which multiple receptor types are expressed will yield results different from tissues expressing only one type, unless a type-selective ligand is used. 

In radioligand binding studies, non-linear Scatchard plots or competition curves that have abnormally steep slopes imply complex binding phenomena, possibly involving multiple receptor types or affinity states. In such cases, the thermodynamic parameters should be separately determined for each receptor type or affinity state. 

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