Equilibrium measurement binding studies

Fluorescence polarization is also well suited to equilibrium binding studies when the free and bound species involved in the equilibrium have different rotational rates (Scheme 5.1). Most molecular interactions can be analyzed by this method. It should be emphasized that, in contrast to other methods using tracers, fluorescence polarization provides a direct measurement of the ratio of bound and free tracer without prior physical separation of these species. Moreover, measure-... 

The interaction of ACh with the Torpedo nAChR. The data shown compare the equilibrium binding parameters obtained from fluorescence studies using covalently attached fluorescent probes and those obtained from radiolabelled [ H]ACh binding studies or functional measurements of cation flux. These data support a model in which the Torpedo nAChR carries sites of different affinities for ACh. We have previously suggested that occupancy of the lower affinity sites leads to channel activation whereas the higher affinity sites may play a role in desensitization processes... 

One disadvantage of frontal analysis is that it requires a relatively large amount of analyte for study. However, frontal analysis does provide information on both the association constant for a solute and its total number of binding sites in a column. This feature makes frontal analysis the method of choice for high accuracy in equilibrium measurements, because the resulting association constants are essentially independent of the number of binding sites in the column. 

A technique commonly employed in binding studies involves equilibrium dialysis in which the concentrations of free and/or bound ligands in equilibrium with the biomacromolecules are measured, where [A,] = [A] -i- [At] ([AJ, [A], and [Ab] are respectively molar concentrations of the total, free and bound ligands). If the total molar concentration of biomacromolecule is [PJ, the number of moles of A bound per mole of P, V is given by... 

A modified (horse) cytochrome c, in which Met-80 is carboxymethylated and therefore unable to coordinate to Fe, was studied by Di Marino et al [145]. At pH 7 the Fe(II) form, by analogy with deoxymyoglobin, is able to bind Oj and CO. Thus the sixth coordination position is vacant or is occupied by weakly bound HjO. With a Au electrode, either as wire or coated onto carbon, but without further modification, cyclic voltammetry was observed with peaks due to reduction and oxidation. There was clear evidence for specific adsorption of oxidized reactant, and the authors did not analyze their results further. The reduction potential was determined to be — 218 mV from equilibrium measurements. 

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