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