Characterization of binding equilibrium data by a variety of fitted isotherms

Abstract

Experimental binding equilibrium data, resulting from measurement of ligand binding to macromolecular carriers, are usually described by fitting of binding constants. These constants are often highly variable, as illustrated in the present paper by two examples, binding of salicylate to human serum albumin and of oxygen to hemoglobin. In order to avoid over-interpretation of binding constants it is pointed out that the best-fit solution, obtained by least-squares fitting, may be supplemented by several, e. g. thirty, acceptable solutions. It is further shown how the 30 sets of acceptable binding constants, plotted as Klotz' affinity profiles, can serve for evaluation of cooperative effects.Multiple binding equilibria for a ligand to a macromolecular carrier are usually characterized by a set of binding constants, parameters in a binding equation. Such a set of binding constants may serve two purposes, comparison with results from other binding equilibrium studies, and considerations of interactions, i. e. whether binding is cooperative, homogeneous or anticooperative. It is essential for these purposes to have a knowledge of the variation of the binding constants, as originating from the random errors of the observed data.The present paper evaluates the variation of multiple binding constants. Two experimental materials are analysed: data for the binding of salicylate anion to human serum albumin, from equilibrium dialysis measurements, and oxygenhemoglobin data, as published by Winslow et al. [l]. It is shown that the data can be presented by a multitude of fitted isotherms with highly variable binding constants. The results are utilized for consideration of binding interactions. MATERIALS AND METHODSHuman serum albumin was obtained from AB Kabi (Stockholm, Sweden) and was defatted with charcoal in acid solution [2] and lyophilized. Binding equilibria with sodium salicylate were studied by equilibrium dialysis as previously described [3] in a 0.1 M Tris/HCl buffer with 0.1 M sodium chloride, pH 8.2, at 25°C. The time needed for establishing cquilihriiiin was about 8 h; 20 h dialysis time was allowed. absorbance in a 0.5-cm cell at the wavelength 296 nm. It was found that the absorbance was proportional to the concentration within the range of concentrations used. 0 -2 mM, showing the absence of ligand dimerisation. The molar absorption coefficient was 3530 M-' cm-'. Albumin concentration was 100 pM. Errors from binding of salicylate to the membrane and from compartment volume changes were found to be negligible as were a number of other sources of technical errors previously discussed [3]. Free and bound equilibrium concentrations of salicylate were calculated and plotted as r, the concentration of bound salicylate divided by that of albumin, versus the logarithm of the free salicylate concentration. Additional points obtained with varying albumin concentrations, from 100 pM to 1000 pM, were apparently located on the same curve, indicating the absence of albumin-albumin interaction.Equilibrium data for binding ...

Thermodynamic parameters for binding of fatty acids to human serum albumin

“…We have accordingly analyzed our data in terms of the general binding equation [24]. In the iterative evaluation of the stoichiometric constants it is possible to account for the stochastic errors on the experimental data [9]. We thus arrive at the conclusion that the affinity of albumin for fatty acids increases with chain length and decreases with temperature, in accordance with previous work by Spector et al [17] and by Ashbrook et al [25,26].…”

“…Calculations of binding constants by an iterative procedure were performed with a Digital PC 350 computer [9].…”

“…Saturation of albumin, however, cannot be achieved 1121, and Nwas given the smallest integer value compatible with optimal fitting of the binding equation to the experimental data. The iterative procedure, as developed by Brodersen et al [9] yields sets of binding constants that fit the observations equally well within a chosen probability limit. A rough estimate of the first stoichiometric constant, K 1 , can be obtained using a simple graphical approach.…”

“…This quantitative analysis will focus on binding data for r < 3. A best-fit approximation was performed by iteration of binding constants in Eqn (1) to this selection of experimental data [9]. The values for the first two stoichiometric constants, giving this best-fit approximation, are shown in Table 1 Table 1 are included the range of 30 acceptable sets of constants within a probability limit of 0.75.…”

Role of Hydration in the Binding of lac Repressor to DNA

“…Molar volumes were calculated according to V mol ϭ v ⅐M r . Error Analysis-95% confidence intervals for fitted parameters were estimated by the method of Broderson et al (38). Timasheff and co-workers (11,15) have shown that many low molecular weight osmolytes are excluded from the volumes immediately adjacent to proteins (i.e.…”