From: jack correia <jcorreia@fiona.umsmed.edu> To : David J. Scott <djs17@york.ac.uk> Date: Wed, 22 Apr 1998 11:20:54 -0500

An addendum to Walter's comments - if the equilibrium constant is large and you are working at 2 orders of magnitude higher concentration, such that the fraction of complex/aggregate in the boundary is > 99.9%, then the area under the curve (if you are using absorbance correct this to moles with an extinction coefficient) is the concentration of complex. If you are looking at a mixed system, like the antibody-antigen system, which has a large equilibrium constant, and you use different stoichiometries, then the area under the complex boundary is all complex and the area under the trailing boundary is the free concentration of the excess component. This is actual what we call stoichiometric binding because "all" of the component not in excess it in complex, ie. there is no free material, or not enough to measure! So under these conditions, very large K, you can not calculate an equilibrium constant by taking ratios of areas. That is unless it is frozen as Walter suggested. For most systems you get an reaction boundary where everything exist and equilibrium is obeyed at each radial position in the cell. For these systems you can try looking at the concentration dependence of the weight average sedimentation coefficient and fit it to an equilibrium constant. You must know the S values of all the complexes, or have reasonable estimates, and you must obviously have a good idea about the stoichiometries or the model to use. -------------------------------------------------| | Dr. John J. "Jack" Correia | | Department of Biochemistry | | University of Mississippi Medical Center | | 2500 North State St. | | Jackson, MS 39126 | | (601) 984-1522 | | fax (601) 984-1501 | | email: jcorreia@fiona.umsmed.edu | | homepage: | | http://fiona.umsmed.edu/~biochem/correia.html | -------------------------------------------------|

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