Patent ID: 8224582

Claim:
A homogenous method for determining the kinetic parameters of a complex (L•T) formed between two components (L and T) wherein such parameters are: (i) the equilibrium dissociation constant, K d , of said complex (ii) the monomolecular rate constant, k off , of the decay of said complex (iii) the bimolecular rate constant, k on , of the formation of said complex wherein the method comprises: a) reacting said components under conditions that promote the formation of said complex, and allowing the reaction to come to equilibrium resulting in an equilibrium mixture; wherein said equilibrium mixture can be prepared inside the capillary or outside the capillary, wherein if said equilibrium mixture is prepared outside the capillary a plug of said equilibrium mixture is introduced into the capillary, wherein said capillary is a part of a capillary electrophoresis instrument; b) subjecting said equilibrium mixture to capillary electrophoresis under non-equilibrium conditions to permit the decay of said complex and separation of said components and complex; c) monitoring the migration of one or more said components and said complex through a detection point on or off the capillary to generate a migration pattern thereof including peaks and curves, the areas under which represent the amounts of said one or more components and/or complex that passed through said detection point; d) determining said equilibrium dissociation constant using one of two following equations or their modifications: K d = A L ⁢ A T A L · T + A decay K d = [ T ] 0 ⁢ ( 1 + A L A L · T + A decay ) - [ L ] 0 1 + 1 / A L A L · T + A decay wherein A L , A T , and A L•T are normalized areas of said peaks corresponding to said components L and T and said complex L•T, respectively; A decay is the area under one said curve corresponding to the decay of said complex L•T during said capillary electrophoresis; [T] 0 and [L] 0 are total concentrations of said components T and L in the equilibrium mixture, respectively; wherein the first equation is applicable to cases when both L and T are detectable and the second equation is applicable to cases when only one said component, L, is detectable; e) determining said monomolecular rate constant by fitting one or more said curves of decay of said complex with a single-exponential function: I t = I t 0 ⁢ exp ⁢ { k off ⁢ t L · T t C - t L · T ⁢ ( t - t 0 ) } or ⁢ ⁢ by ⁢ : k off = ln ⁡ ( ( A L · T + A decay ) / A L · T ) t L · T wherein t 0 and t are initial and variable time points on said curve of decay of said complex, t C is the migration of one of said components, whose signal constitutes said curve, and t L•T is the migration time of said complex in said capillary electrophoresis; f) determining said bimolecular rate constant by: k on =k off /K d .