Human Serum Albumin Immobilized On Magnetizable Beads: A Rapid Method for Compound HSA Binding Study

Abstract

Immobilized human serum albumin (HSA) was developed by coupling His-tagged HSA onto Ni2+-coupled magnetizable beads (HSA-beads), allowing the HSA to be easily removed from incubation components. The HSA-beads system provides a rapid and convenient method to study HSA compound binding. In this study, the HSA-beads system was characterized and evaluated as a tool for assessing compound HSA binding properties. The free fraction (fu) values of test compounds measured using HSA-beads were comparable to those determined by equilibrium dialysis (ED), which is commonly used to evaluate albumin binding in vitro. The equilibrium dissociation constant (Kd) values determined for a series of compounds using the HSA-beads method demonstrated good correlation with literature data. This good correlation also suggests that the binding of His-HSA to the beads does not impact the conformations of the two compound binding sites of HSA, as the range of compounds tested encompassed binding to both sites. Furthermore, the Kd values of representative compounds itraconazole and BIRT2584 that were difficult to assess using ED, due to significant cellulose membrane adsorption, were successfully determined. The HSA-beads provide several advantages over ED, such as simple preparation, short assay incubation duration, and the ability to quantify both free and HSA-bound species of the test compound, facilitated by the simple separation of HSA-beads from the solution phase using a magnetic field. These properties render the HSA-beads method suitable for high-throughput studies on compound HSA binding.