5 and 1 is considered to be representative of a high quality assay this example illustrates thermodynamic characterization of tnyl raw peptide binding to ephb4 ligand binding domain ephb4 lbd in these experiments we monitored the binding of ephb4 lbd to ephrin b2 and peptide ligands using isothermal titration calorimetry itc the interaction between ephb4 17 196 and ephrin b2 yielded a kd of 40 nm and a u03b4ho of 3 3 kcal mol 1 this is slightly lower than the affinity reported for the interaction between the entire mouse ephb4 extracellular domain and mouse or human ephrin b2 without being limited by theory we hypothesize that the difference may be explained by the existence of a third low affinity eph ephrin interface located outside the ephrin binding domain smith f m et al j biol chem 279 9522 9531 2004 in addition n and c terminal truncations of the peptide as well as targeted mutations in the center of the peptide were synthesized in order to biophysically validate individual effects of the peptide upon ephb4 binding table 5 presents data of a thermodynamic analysis of wtephb4 and mutant ephb4 binding to ephrin b2 and tnyl raw and related peptides the table shows the results of isothermal titration calorimetry itc analysis the kd values reported from this method compare well with the kd values determined from the fp assays fig6 and 7 three regions of interactions proved critical for receptor binding the n terminal tyr the phe.