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 source of error in sedimentation coefficients obtained by sucrose density gradient centrifugation of proteins undergoing reversible self-association. The error stems from the excluded volume (molecular crowding) effect of the sucrose on the activity coefficients of monomeric and polymeric 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 coexistence of monomer if 5% sucrose suffices to displace the equilibrium completely 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 migrated into the sucrose gradient. These features are illustrated by the results of computer-simulated sedimentation of reversibly dimerizing systems in a sucrose gradient, and by conventional sedimentation velocity experiments on yeast enolase.
Cann
J R
JR
Department of Biochemistry/Biophysics/Genetics, University of Colorado Health Sciences Center, Denver 80262.
Coombs
R O
RO
Howlett
G J
GJ
Jacobsen
M P
MP
Winzor
D J
DJ
D002501:Centrifugation, Zonal / Q000706:statistics & numerical data*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D046911:Macromolecular Substances; D015735:Mifepristone / Q000378:metabolism; D010751:Phosphopyruvate Hydratase / Q000737:chemistry; D011397:Promegestone / Q000378:metabolism; D011506:Proteins / Q000737:chemistry*; D011980:Receptors, Progesterone / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013395:Sucrose / Q000494:pharmacology
D046911:Macromolecular Substances; D011506:Proteins; D011980:Receptors, Progesterone; D015735:Mifepristone; D013395:Sucrose; D011397:Promegestone; D010751:Phosphopyruvate Hydratase