Characterization of weak protein dimerization by direct analysis of sedimentation equilibrium distributions: the INVEQ approach.

Closer scrutiny has been accorded a recently reported procedure for characterizing weak protein dimerization by sedimentation equilibrium (INVEQ) in which the equilibrium distribution is analyzed as a dependence of radial distance on solute concentration rather than of solute concentration on radial distance. By demonstrating theoretically that the fundamental parameter derived from the analysis is simply the difference between the dimerization constant and the osmotic second virial coefficient for monomer-monomer interaction, this investigation refutes the original claim that independent estimates of these two parameters can be obtained by nonlinear curve fitting of the sedimentation equilibrium distribution. This criticism also applies to conventional analyses of sedimentation distributions by the commonly employed Beckman Origin and NONLIN software. Numerically simulated distributions are then analyzed to demonstrate limitations of the procedure and also to indicate a means of improving the reliability of the returned estimate of the dimerization constant. These features are illustrated by applying the original and revised analytical procedures to a sedimentation equilibrium distribution for alpha-chymotrypsin (pH 4.0, I 0.05 M).