Ultrafast affinity extraction with non-covalent entrapment for biointeraction analysis: development and applications to single- and multi-site drug-protein binding.

Reversible biomolecular interactions play a vital role in numerous biological processes. One of the techniques that has been developed to evaluate solution-phase binding and rate constants for such processes is ultrafast affinity extraction (UAE). In UAE, a microcolumn containing an immobilized capture agent is used to quickly remove the free form of a target solute from a sample that also contains a soluble binding agent. This separation is typically done on the millisecond-to-second time scale. The measured level of the captured fraction can then be utilized to estimate the binding and rate constants for the interactions between the solute and binding agent in the sample. Covalent coupling methods have been used in the past with UAE to immobilize serum proteins as capture agents for various drugs. This work examines the combination of non-covalent entrapment to immobilize human serum albumin (HSA) as a capture agent within microcolumns that are then used in UAE to study drug interactions with soluble HSA. This combined approach is evaluated using racemic warfarin and repaglinide as model drugs with known single-site or multi-site binding to this protein. The theoretical and experimental behavior of UAE is examined in each case. The binding and rate constants determined by this method are also compared to reference values, including results obtained by UAE using a covalently immobilized capture agent. The results are then used to identify the potential strengths or limitations of this approach and to provide guidelines on how this method can be applied in the future to examine other types of biointeractions.