EFFECTS OF MOLECULAR CROWDING ON PROTEIN SELF-ASSOCIATION - A POTENTIAL SOURCE OF ERROR IN SEDIMENTATION COEFFICIENTS OBTAINED BY ZONAL ULTRACENTRIFUGATION IN A SUCROSE GRADIENT

Theoretical and experimental studies have illustrated a potential sour ce of error in sedimentation coefficients obtained by sucrose density gradient centrifugation of proteins undergoing reversible self-associa tion. The error stems from the excluded volume (molecular crowding) ef fect of the sucrose on the activity coefficients of monomeric and poly meric states. The consequent displacement of the equilibrium position in favor of polymeric state(s) is a function of sucrose concentration, and can therefore result in failure to detect the equilibrium coexist ence of monomer if 5% sucrose suffices to displace the equilibrium com pletely toward dimer. In less extreme situations, it may result in the evaluation of an average sedimentation coefficient whose magnitude is a function of sucrose concentration and hence of the distance migrate d into the sucrose gradient. These features are illustrated by the res ults of computer-simulated sedimentation of reversibly dimerizing syst ems in a sucrose gradient, and by conventional sedimentation velocity experiments on yeast enolase.