Quantitative analysis of Ca(2+)-binding by flow dialysis.

Abstract

Ca2+-binding to proteins can be measured directly by equilibrium dialysis (,), the standard method for the direct measurement of the binding of small ligand molecules by macromolecules. In this method, a semipermeable cellulose bag containing a solution of macromolecules is immersed in the buffer solution containing ligand molecules and is incubated to attain both the chemical and diffusion equilibrium. The method can be improved with the use of two small thin chambers separated by the cellulose membrane, which may reduce the incubation time required to achieve diffusion equilibrium (microdialysis) (). Ligand molecules are usually labeled with the radioactive isotopes for quantitative determinations, and ligand molecules bound to the macromolecule in the equilibrium state are determined directly from the difference between the free concentration in the dialysate and the total concentration in the protein solution. Binding of ligand to the protein molecule can be calculated from the known value of the protein concentration, and the ligand bindings at several free concentrations of the ligand are determined from independent experiments to yield a ligand binding curve from which the maximum number of ligand binding and the equilibrium constants are estimated. In this method, the ligand binding equilibrium, which is usually obtained within less than a second, has to be assessed after attainment of the diffusion equilibrium of ligands across the membrane, which usually takes a much longer time - on the order of several hours.