Determining the kinetic rate constants (ka and kd) and the equilibrium dissociation constants (KD) for small molecule-protein interactions is an important application for label-free instrumentation in the drug discovery and diagnostic industries. Often, small molecule-protein interactions can be difficult to regenerate on biosensor surfaces. In those cases where no appropriate regeneration condition can be found, alternate biosensor methods of kinetic analysis are required. In this application note, we demonstrate a technique referred to as Simulject that requires no surface regeneration. In a Simulject experiment, two concentrations of analyte are injected simultaneously and independently across two flow cells in a BiOptix 404pi biosensor. Previously, it has been shown that two appropriately chosen concentrations of analyte are all that is required to determine the binding parameters for an interaction (1).
Carbonic anhydrase II from bovine erythrocytes (Sigma) was amine-coupled to an average surface capacity of 4100 RU (+1.5%) on two flow cells of a BiOptix CMD200 sensor chip using standard EDC/NHS coupling chemistry with blocking of the remaining active ester groups by ethanolamine. Two reference flow cells were activated and blocked with no immobilized protein. The running buffer was PBS, 0.05% polysorbate 20, pH 7.4. Two concentrations of 4-(aminomethyl)benzenesulfonamide (ABS) (Fluka) were injected in duplicate at 100 μL/min at 20°C as indicated in Table 1. Five buffer blanks were injected for “warming up” the surface and double-referencing before the ABS injections. Figure 1 illustrates schematically the Simulject experiment.
BiOptix 404pi biosensor run in 2×2 mode
Data were collected on the BiOptix 404pi instrument and were processed using Scrubber 2.0c (Biologic Software). The data were globally fit from two different flow cells with nearly identical surface capacities of CA II to a 1:1 interaction model as shown in Figure 2. Table 2 shows the fitted ka ,kd and KD values by the Simulject method versus a traditional biosensor experiment with five different concentrations of ABS injected.