Analysis of binding

The two important equilibrium questions about a binding process are how many (or stoichiometry) and how tight (or affinity). Information about these quantities can usually be derived from appropriate titrations which enable the concentrations of the interacting partners and complexes to be measured. Let us consider the simplest case of two species A and B which interact to form a single complex C. 

Provided that the free concentrations of reactants and complex are known, by any suitable experimental procedure, the association constant can be determined and thus the affinity of the interaction. 

The situation often arises, for example in the binding of a ligand to a cell surface receptor, where not even the stoichiometric concentration of one of the components is known. The total concentrations of ligand and cells may be known, but the number of receptors per cell (and hence the total concentration of receptor) is often not known, and this is one of the parameters that needs to be evaluated. 

The mass action relationship in this case can be written in the form  

In deriving this equation it has been assumed that all of the receptors have the same affinity and bind independently at one another. Under these conditions, Eqn. 8.19 can be re-formulated as a linear equation 

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Figure 4 shows an analysis of binding and dissociation of FLPEP in permeabilized cells in the absence (top panel) and the presence of GTPyS (lower panel). Dissociation is initiated, as indicated, by the addition of a receptor antagonist, tBoc-phe-leu-phe-leu-phe. The solid line is a fit to the data. The data from this experiment have... 

Computational analysis of binding site interactions is achieved by calculating the relative binding free energy (AAGbind) for a ligand L with an... 

A first approach to study the interaction of posttranslational modified Ras proteins with membranes was the analysis of binding and exchange of isoprenyl-ated peptides with and between lipid vesicles utilizing a fluorescent bimanyl label. Studies with K-Ras peptides revealed that a single isoprenyl group is sufficient for membrane association only if supported by carboxymethylation of the C-terminal cysteine [227,228]. 

Kinetic studies were attempted at 4, 15, 25 and 37 °C, but the colloids tended to aggregate at all temperatures above 4°C. Four models were used to determine the binding mechanisms from the kinetic data. A detailed analysis of binding at 4 °C, was made. Models were set up involving one or two surface sites which also satisfied the overall kinetics but the analyses were not definitive. Although it was demonstrated that the cells were capable of endocytosis of fluorescence-conjugated transferrin, there was no evidence for the endocytosis of the cationic colloids. 

Most laboratories now have access to powerful computers and an extensive array of commercially available data analysis software (e.g., Prism (GraphPad, San Diego, CA), Sigma Plot (San Rafael, CA)). This provides ready access to the use of nonlinear regression techniques for the direct analysis of binding data, together with appropriate statistical analyses. However, there remains a valuable place for the manual methods, which involve linearisation, particularly in the undergraduate arena and these have been rehearsed in the above text. 

Premises Underlying SCAM Analysis of Binding Site Structure. 441... 

A number of useful reviews of small molecule-protein interaction have appeared (D2, E4, S26, W7), and these contain detailed treatments of the mathematical analysis of binding data as well as information on experimental methods. 

Analysis of binding experiments required a careful comparison of (i) the MYKO 63 bands, either in the presence or absence of DNA bands and (ii) the DNA Raman bands, either in the presence or absence of MYKO 63 bands. This comparison was achieved by computer-subtracting variable amounts of one spectrum from another. Previously, the various spectra were normalized to the same relative Raman intensity, with the 934 cm band (CIO symmetric stretch) as an internal standard. The intensity of the CIO. scattering measures the combined effect of such experimental factors as counting time, optical alignment and laser power. 

NHH Heegard, RT Kennedy. Identification, quantitation and characterization of biomolecules by capillary electrophoretic analysis of binding interactions. Electrophoresis 20 3122-3133, 1999. 

The more detailed analysis of binding of AREF to the ARE is shown in... 

Roelofs, W.G.H., "Thin-layer chromatography, an aid for the analysis of binding materials and natural dyestuffs from the work of art ICOM Plenary Meeting Madrid, Oct. 1972. 

Both equations are used in the linear and graphical analysis of binding data. 

From a stereochemical analysis of binding anions, the interlocking-sites model is most consistent with available data. We have approached the question of the spatial relationship of metal ion and anion in human transferrin through electron and nuclear magnetic resonance spectroscopy (17,49). 

With 3D structures of receptor-ligand complexes at hand, the analysis of binding phenomena can of course be much more detailed. The binding affinity AGbinding can be estimated as a sum of interactions multiplied by weighting coefficients AG,-... 

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