Effect of Kinetics on Sedimentation Velocity Profiles and the Role of Intermediates

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We have previously presented a tutorial on direct boundary fitting of sedimentation velocity data for kinetically mediated monomer-dimer systems (Correia & Stafford, 2009). We emphasized the ability of Sedanal to fit for the koff values and measure their uncertainty at the 95% confidence interval. We concluded for a monomer-dimer system the range of well determined koff values is limited to 0.005 to 10−5 sec−1 corresponding to relaxation times of ∼70 to ∼33000 sec. 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 (K1 = 104M−1, K2 = 108 M−1). Under these conditions a k1,off < 0.01 sec−1 produces slow kinetic features. The low concentration of species C contributes to this effect while still allowing the accurate estimation of k1,off (although k2,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 with partial resolution of individual species during sedimentation. Cooperativity of ring formation drives the reaction and thus separation of kinetics and energetics can be challenging. This situation is experimentally exhibited by systems that form large oligomers or rings, formation of micelles and various protein aggregation diseases including formation of β-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.