Binding parameters

F ure 8J. Detenninaiion of the slc iomary of the TN Q aci(l g ycoprotein complex at 20 C with the Job plot ([TNS] + ([([ai-acid glycoprotein] = 4.5 pM. Source Altani, J. Vos, R, Willaert. K and Engdbor is. V. 1995. Pholochem. PhotobioL 62, 30-M. AuAorteatloo of reprint accorded the American Society for Photochemistiy 

The standard enthalpy change for the binding is A F = -18 3 kJ mol and AS =19 12 J K mol The negative value of the enthalpy change indicates that the interaction between TNS and a i-acid glycoprotein is exothermic and is not of a pure nonhydrophobic nature. This means that while TNS binds to a hydrophobic site, it is in close contact with the buffer. 

Hgure 8.7. Steady-state fluorescence spectra of llie 2,6-TNS-ai-ocid glycoprotein complex recorded at four e. cHation wavelenglhs, 360 nm (a), 370 nm (b), 380 nin (c) and 400 im (d). [TN =- [tti-acid coprotein] - S pM, temperature 20°C Source Albani, J. 1992, Biopl Chem.44, 129-137. 

The behavior of the plot = ) is tentperature independent This result 

We notice that emission maximum does not present the same position at the three temperatures. I lowever. this change is not regular along the excitation wavelengttis. For instance, at 335 nm and 340 nm, the difference of cmis on maxima at 8 C and 40 is more than 30% of full range emission maxima changes, while at 320 and 395 nm. the difference is much less important. 

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As Fig. 16 shows, the preferential binding of DMSO, DMF and NMF from aqueous solution to (Lys HBr)n at low contents of the organic solvent x increases with its concentration. However, at approximately x3 = 0,2 a maximum is reached and then preferential hydration between x3 = 0,3 and 0,5 occurs. No preferential binding was observed for NMP, EG or 2 PrOH, however increasing hydration occured with x3. Only in 2 PrOH at x3 > 0,3 a-helix formation occured. Furthermore binding parameters for the systems NMP + DMSO, EG + DMSO and DMF + DMSO have been determined. An initial preferential binding of DMSO by (Lys HBr)n, a maximum and a subsequently inversion of the binding parameter was also observed in these mixtures. The order of relative affinity is DMSO > DMF > EG > NMP. In DMF/DMSO-mixtures (Lys HBr) attains an a-helical conformation above 20 vol.- % DMF and in 2-PrOH/water above 70 vol.- % 2 Pr-OH. 

Waldmann-Meyer, HK, Protein Ion Equilibria, Total Evaluation of Binding Parameters and Net Charge from the Electrophoretic Mobility as a Function of Ligand Concentration. In Recent Developments in Chromatography and Electrophoresis Frigerio, A McCamish, M, eds. Elsevier Scientific Amsterdam, 1980 Vol. 10, p 125. 

Table 3 Various binding parameters for the interaction of berberine with several DNAs [144,161] ...

Table 8 Binding parameters of t-RNA complexation with berberine and palmatine obtained from spectrophotometric, spectrofluorimetric and isothermal titration calorimetric study [214]... 

L. G. Reevaluating equilibrium and kinetic binding parameters for lipophilic drugs based on a structural model for drug interaction with biological membranes. 

Mason, R. R Rhodes, D. G. Herbette, L. G., Reevaluating equilibrium and kinetic binding parameters for lipophilic drugs based on a structural model for drug interaction with bilogical membranes, J. Med. Chem. 34, 869-877 (1991). 

Combined use of Eqs. (43)—(45) allows free drug concentrations to be predicted for each subcompartment. This approach to modeling free drug concentrations would make use of protein binding parameters (i.e., Bt, Kt) obtained from in vitro experiments. 

Protein binding parameters B, and Kh as depicted in Eq. (44), are obtained from in vitro experiments utilizing filtration, centrifugation, and dynamic dialysis or equilibrium dialysis methods. These techniques have been reviewed elsewhere [54,55],... 

Daum, P. R., Hill, S. J. Young, J. M. (1982). Histamine Hl-agonist potentiation of adenosine-stimulated cyclic AMP accumulation in slices of guinea-pig cerebral cortex comparison of response and binding parameters. Br. J. Pharmacol. 77, 347-57. 

T. K. Dam, R. Roy, D. Page, and C. F. Brewer, Thermodynamic binding parameters of individual epitopes of multivalent carbohydrates to concanavalin A as determined by reverse isothermal titration microcalorimetry, Biochemistry, 41 (2002) 1359-1363. 

F. V. Bright, T. L. Keimig, and L B. McGown, Thermodynamic binding parameters evaluated by using phase-resolved fluorescence spectroscopy, Anal. Chim, Acta 175, 189-201 (1985). 

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... 

The above discussion provides only a brief overview of how fluorescence techniques can be used to study the interactions of ligands with their receptors. We have focused on the quantitation of the binding parameters and compared the data with that which may be obtained with those from radiolabelled ligand binding studies. The number of applications of fluorescence in the study of neurochemistry and molecular biology is ever increasing. Outside the scope of this review is, for example, the use of fluorescence microscopy to monitor cell surface expression and targeting of receptors or the use of fluorescence probes to monitor ion transport into and out of cells. 

The appropriate definition of nonspecific binding is essential prior to characterization of the kinetic and equilibrium properties of the binding interaction. As a rule, nonspecific binding can be defined using a concentration of the unlabelled ligand that is 100 times its Ka value for the sites of interest. Failure to appropriately define nonspecific binding will invalidate the determination of the binding parameters. 

In the absence of crystallographic data, there exist many other experimental criteria that allow the determination of the binding mode [30,38]. They can be classified into two categories the measurement of physical effects on DNA and spectroscopic studies (Table 1, first column, C and A respectively). Irrespective of the binding mode, the binding parameters (the affinity constant and the number n of occupied base pairs per molecule) can be determined from equilibrium dialysis and Scatchard plots [43 46]... 

Crisponi, G., Nurchi, V., and Ganadu, M.L. (1990), An Approach to Obtaining an Optimal Design in the Non-Linear Least Squares Determination of Binding Parameters in a Complex Biochemical System, J. Chemom., 4, 123-133. 

R8. Rosenthal, H. E., A graphic method for the determination and presentation of binding parameters in a complex system. Anal. Biochem. 20, 525-532 (1967). 

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