The use of diafiltration for the measurement of dialysable ligand binding to macromolecules: an examination of the theoretical parameters using concanavalin A in an Amicon cell

Abstract

The technique of diafiltration as a means of determining the characteristics of small ligand binding by proteins has been evaluated by measuring the binding of 45Ca to concanavalin A. Deposition of protein on the diafiltration membrane was found to be an important source of error but which could be eliminated by presaturation of the membrane with polylysine. The association constant for concanavalin A and calcium was 1.8 × 104 M−1, with a value of n = 2.2 atoms of calcium being bound per dimer of concanavalin A.The concentration of protein relative to the dissociation constant is also an important variable, and it was found that the method of diafiltration is only useful when protein concentrations greater than the value of the dissociation constant are used.