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

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 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 0.005–10−5s−1 corresponding to relaxation times of ~70 to ~33,000s. 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=108M−1). Under these conditions a k1,off