Human versus Bovine Serum Albumin: A Subtle Difference in Hydrophobicity Leads to Large Differences in Bulk and Interface Behavior

The protein human serum albumin (HSA) is able to readily crystallize in the presence of trivalent cations, whereas this is not the case for the homologous protein in cattle, bovine serum albumin (BSA), although both have analogous functions as well as similar physicochemical properties. To understand the underlying interactions and mechanisms, we investigated their bulk phase behavior with CeCl3 by visual inspection, optical microscopy, and small-angle X-ray scattering (SAXS). The results reveal that both proteins undergo reentrant condensation and liquid–liquid phase separation (LLPS). However, the LLPS binodal for HSA shifts toward lower protein concentrations than that for BSA, indicating a stronger intermolecular attraction in HSA solutions at the same compositions, consistent with SAXS measurements. Moreover, crystallization occurs within the condensed regime of HSA, but no crystallization was observed for BSA. Adsorption studies at a hydrophilic SiO2 surface demonstrate that both systems show reentrant adsorption with a higher amount of adsorbed BSA, likely due to enhanced cation-mediated interactions and/or hydrogen bonds. We conclude that the higher surface hydrophobicity of HSA could explain the experimental observations. These additional hydrophobic interactions not only strengthen the attraction between the proteins but also provide directional and specific protein–protein contacts, which are favored for protein crystallization. This work further demonstrates the sensitivity and complexity of protein interactions in solution: subtle differences in molecular structure lead to a dramatic change in their phase behavior. Generalization of these findings can pave the way toward, e.g., better drug design and improve medical treatment.