title: Effect of kinetics on sedimentation velocity profiles and the role of intermediates.
abstract: We have previously presented a tutorial on direct boundary fitting of sedimentation velocity data for kinetically mediated monomer-dimer systems [Correia and Stafford, 2009]. We emphasized the ability of Sedanal to fit for the k(off) values and measure their uncertainty at the 95% confidence interval. We concluded for a monomer-dimer system the range of well-determined k(off) values is limited to 0.005-10(-5) s(-1) corresponding to relaxation times of approximately 70 to approximately 33,000 s. More complicated reaction schemes introduce the potential complexity of low concentrations of an intermediate that may also influence the kinetic behavior during sedimentation. This can be seen in a cooperative ABCD system (A+B --> C; B+C --> D) where C, the 1:1 complex, is sparsely populated (K(1)=10(4) M(-1), K(2)=10(8) M(-1)). Under these conditions a k(1,off)<0.01 s(-1) produces slow kinetic features. The low concentration of species C contributes to this effect while still allowing the accurate estimation of k(1,off) (although k(2,off) can readily compensate and contribute to the kinetics). More complex reactions involving concerted assembly or cooperative ring formation with low concentrations of intermediate species also display kinetic effects due to a slow flux of material through the sparsely populated intermediate states. This produces a kinetically limited reaction boundary that produces partial resolution of individual species during sedimentation. Cooperativity of ring formation drives the reaction and thus separation of these two effects, kinetics and energetics, can be challenging. This situation is experimentally exhibited by systems that form large oligomers or rings and may especially contribute to formation of micelles and various protein aggregation diseases including formation of beta-amyloid and tau aggregates. Simulations, quantitative parameter estimation by direct boundary fitting and diagnostic features for these systems are presented with an emphasis on the features available in Sedanal to simulate and analyze kinetically mediated systems.
authors.affiliation: Department of Biochemistry, University of Mississippi Medical Center, Jackson, Mississippi, USA.
authors.forename: John J
authors.initials: JJ
authors.lastname: Correia
authors.affiliation: Department of Biochemistry, University of Mississippi Medical Center, Jackson, Mississippi, USA.
authors.forename: P Holland
authors.initials: PH
authors.lastname: Alday
authors.affiliation: Boston Biomedical Research Institute, Watertown, Massachusetts, USA.
authors.forename: Peter
authors.initials: P
authors.lastname: Sherwood
authors.affiliation: Boston Biomedical Research Institute, Watertown, Massachusetts, USA.
authors.forename: Walter F
authors.initials: WF
authors.lastname: Stafford
mesh_terms: D000465:Algorithms; D002498:Centrifugation; D003198:Computer Simulation; D007700:Kinetics; D008958:Models, Molecular