Binding constants and stoichiometry

Rudnev, A.V., Aleksenko, S.S., Semenova, O., Hartinger, C.G., Timerbaev, A.R., Keppler, B.K. Determination of binding constants and stoichiometries for platinum anticancer drugs and serum transport proteins by capillary electrophoresis using the Hummel-Dreyer method. J. Sep. Sci. 28, 121-127 (2005)... 

A similar strategy has been applied by Mazik and coworkers. The receptor is usually titrated with increasing amounts of the sugar, to evidence binding, to extract the values of the binding constants and to get the stoichiometry of the complexes formed. Reverse titrations and extraction experiments can also be performed. The structures of the receptors described are based on different scaffolds the above mentioned hexasubstituted tripodal benzene moiety and 3,3, 5,5 -tetrasubstituted... 

To validate the method, we applied PLIMSTEX to determine the binding constants (Xa), stoichiometry (1 protein to n ligands), and the protection against H/D exchange in various interactions. We chose as tests the binding of Mg to gua-nosine diphosphate (GDP)-bound human ras protein, of Ca + to apo calmodulin... 

Therefore two main arrangements are applied. For a slow rate of dissociation related to the CE run-time scale the sample can be preincubated prior to electrophoresis and the reaction set to equilibrium. After equilibrium is obtained, an aliquot of the reaction mixture is analyzed by CE, which is used simply as a method to separate and quantify the analyte, ligand, and complex in the preequilibrated mixture. During the CE run, which is carried out in a buffer without reactants, no equilibrium is established. However, as the rate of dissociation is comparably slow, the reactants and the complex present in the mixture can be separated and their concentrations (which are those of the incubation equilibrium) determined quantitatively. Change of the mutual concentrations of analyte and ligand in the mixture allows conclusion about the binding constant and the stoichiometry of the complex formed. 

After a decade of intensive development, ACE is widely recognized nowadays as a powerful tool in the study of different kinds of biomolecular interactions. More than 400 scientific reports related to ACE can be found in the literature, covering almost all fields of bioanalytical chemistry. Unique features of homogeneous analysis coupled with the separation power of CE makes ACE especially favorable for precise determination of affinity parameters, such as binding constants and binding stoichiometries. Automation, multicapillary arrows, and chip technology increase throughput of ACE analysis, a factor which still limits... 

The most thoroughly studied host-guest system with a porphyrin-based host is the complex formed between amines and metalloporphyrins. Tire stoichiometry of the host-guest complex and the binding affinity will depend on the nature of the metal, the basicity of the amine, the reaction media and the pH in the case of water. Porphyiin-amine equilibria in water were studied by Pasternack et u/. [5.10,15,20-tetrakis(/V-methylpyridinium-4-yl)porphyrina-tolCo(IIl) (27) binds pyridine in water, with the binding constants and the rale constants of complex formation depending upon pH. At acidic pH, pH < 4, the first replacement of water by pyridine. 

The use of SPR for the measurement of binding parameters, mainly in biological analysis, has been reported. These parameters include reaction kinetics (fea, fed), binding constants and determining the active concentration of molecules. When experiments are performed carefully, SPR biosensors can also be used to determine the binding stoichiometry and mechanism of the interaction [21-26]. 

The data for binding constants of the systan are directly available without knowing molar absorptivity of complexed L or possible absorption by M, since the total ligand concentration in the original sample is independently known. By repeating frontal analysis in different concentrations of L, the binding ratio and stoichiometry is obtained (see equation 2). 

The simplest of all SCM models uses the nonelectrostatic model (NEM), which considers surface equilibria strictly as chemical reactions, without explicit correction for electrostatic attraction or repulsion. In the NEM, the pH-dependent coulombic energy contribution to the mass action equation is included within the apparent binding consliinl. The tippjirent binding constants and the stoichiometry... 

Our work exploited the reported polarity-sensitive fluorescence of curcumin to allow for accurate determinations of host-guest stoichiometries, binding constants, and solubility enhancements for its inclusion into the three parent CD as well as their HP-modified derivatives [94]. We were able to demonstrate... 

Fig. 12. Ras in complex with GppNHp at 600 pmol/1 is injected into a solution of 45 pmol/1 RalGDS-RBD. In the upper panel the (peakwise) change of the heating power is recorded which is necessary to keep the cell at constant temperature after each injection. The integrated peaks of the upper panel are plotted vs the molar ratio of Ras GppNHp/RalGDS in the lower panel. The fitted curve yields the data in the box, where N indicates the stoichiometry, K the affinity constant and H the enthalpy of binding...

The second and third relaxation processes were coupled, where the observed rate constants differed by a factor of 3 to 7 and the rate constant for each relaxation process varied linearly with the DNA concentration.112 This dependence is consistent with the mechanism shown in Scheme 2, where 1 binds to 2 different sites in DNA and an interconversion between the sites is mediated in a bimolecular reaction with a second DNA molecule. For such coupled kinetics, the sum and the product of the two relaxation rate constants are related to the individual rate constants shown in Scheme 2. Such an analysis led to the values for the dissociation rate constants from each binding site, one of the interconversion rate constants and the association rate constant for the site with slowest binding dynamics (Table 2).112 The dissociation rate constant from one of the sites was similar to the values that were determined assuming a 1 1 binding stoichiometry (Table 1). 

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