Equilibrium binding constants determination

The equilibrium binding constant for this 1 1 association is Xu = ki/lLi. The Xu values were measured spectrophotometrically, and the rate constants were determined by the T-jump method (independently of the X,j values), except for substrate No. 6, which could be studied by a conventional mixing technique. Perhaps the most striking feature of these data is the great variability of the rate constants with structure compared with the relative insensitivity of the equilibrium constants. This can be accounted for if the substrate must undergo desolvation before it enters the ligand cavity and then is largely resolvated in the final inclusion complex. ... 

In some instances, flow cytometry assays are a superior alternative to conventional procedures for the determination of equilibrium binding constants (Stein et al., 2001). In contrast to assays that employ radiolabelled ligands, which measure population mean values for binding constants, flow cytometry methods can measure those values in individual cells, revealing heterogeneity in receptor expression within a population of cells or membrane vesicles. Furthermore, small samples can be characterized in a short period of time (hours). This approach to receptor-binding analysis may be limited only by the availability of a properly characterized fluorescent ligand. 

The values of the binding constants determined with different salt concentrations by equilibrium dialyses [43, 48], luminescence titrations and electrochemiluminescence [82], are all 2 or 3 orders of magnitude lower than for ethidium bromide. Therefore, a priori, they do not indicate contribution of classical intercalation into DNA as described for organic molecules and for the DPPZ, HAT and PPZ complexes. 

The repeated pulse injection method was applied to determine the association rate constant of the antigen-immobilized monoclonal antibody reaction 124.25). These monoclonal antibodies differ by their specificity and affinity for HSA. The epitope recognized by the HA6 antibody is located between residues I and 128. The epitope of the commercial antibody (m-anti-HSA) is located between residues 124 and 298 of the HSA molecule. They differ by their affinity for HSA. with equilibrium binding constants of respectively 6.7 x 108 mol-dm and 1.6 x 10K dm3 mol-1 for the HSA/HA6 and the HSA/m-anti-HSA association in solution 25). Close binding rate constants are obtained on both immobilized monoclonal antibodies with Aa = 2.5 x I0 i dm -moC -s-1. 

In another approach Gauthier et. al. (3) devebped a technique based on fluorescence quenching to determine the equilibrium binding constants of polycyclic aromatic hydrocarbons (PAH) with DOC. PAH are eflBcient fluorophores, nonionic, and are usually considered to be insoluble in water. These last two properties thermodynamically drive the PAH from the aqueous phase to the less polar DOC. Also, PAHs are important exan les of hydrophobic organic contaminants in the environment because these properties cause them to accumulate in the l id deposits of higher organisms. 

O Shannessy, D. 1. Brigham-Burke, M. K., S. K. Hensley, P. Brooks, I., Determination of rate and equilibrium binding constants for macromolecular interactions using surface plasmon resonance Use of nonlinear least squares analysis methods. Anal. Biochem. 1993, 212, 457—468... 

O Shannessy, D. J. (1994) Determination of kinetic rate and equilibrium binding constants for macromolecular interactions A critique of the surface plasmon resonance literature. Curr. Opin. Biotechnol. 5, 65-71... 

Therefore, equilibrium binding constants such as or indeed fQ can be determined from a simple ratio of rate constants. Equations such as (7.57) are a form of Haldane relationship, although the term Haldane relationship is more usually applied to expressions in reaction kinetics that link rate constants with an equilibrium constant (see Chapter 8). If the receptor R is immobilised (imm) for any reason by covalent attachment to a solid phase, then the... 

We reasoned that these properties could also be used for the preparation of surface-attached molecular assemblies. For instance, host 1 forms an inclusion complex with the bis (thiol) hydroquinol derivative 2 due to the Tt-donor character of the aromatic nucleus of this guest. The equilibrium binding constant was determined to be 253 + at 25 °C in acetonitrile [10]. When it is... 

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