Determination of dissociation constants between single-protein and multicomponents by a multi-chamber membrane...

The study of single-target multicomponent interactions has long suffered from a lack of effective research methods, primarily relying on the dissociation constant (K_D) determination and competitive displacement models. However, the information obtained is relatively limited and not fully reflects in vivo binding. The developed multi-chamber membrane separation electrophoresis system (MCMSE) maintains the native state of protein coupled with the chromatographic quantification of bound components, it allows for the direct competition method to study single-target multicomponent interactions, effectively complementing the competitive displacement method used in fluorescence-based assays.

As a study case, the multicomponent interactions of tyrosinase (TYR) with kojic acid (KA), fumaric acid (FA), and apigenin (API) were investigated. For the system of TYR with KA and FA, the results of MCMSE and fluorescence spectroscopy (FS) are consistent. Although KA exhibited slightly stronger binding to TYR than FA, FA demonstrated an allosteric competitive effect against KA, with the results of impairing KA binding to TYR. The system of TYR binding to KA and API generated divergent results under the two investigation methods. FS only indicated a significant increase of K_D (TYR-KA) due to the allosteric effect induced by API. In contrast, the MCMSE showed a notable increase in the K_D(s) for both TYR-KA and TYR-API. The two sets of results indicate the possibility of both allosteric effects and binding kinetics jointly determine the binding affinities of KA and API, along with the more intricate conformational changes of TYR.

In complex allosteric systems, a one-way research strategy may provide limited information or even obscure key characteristics of the binding process. The MCMSE serves as a complementary tool applied in direct competition assays for determining K_D (s) of single-target-multicomponent interactions, providing or reflecting more binding processual information in complex systems.