Holographic Fingerprinting Reveals Oligomer-Driven Phase Separation in Bovine Serum Albumin.

Biomolecular condensates formed through phase separation are fundamental to the cellular organization. Although the physical principles underlying the phase separation of intrinsically disordered proteins are well understood, the molecular determinants of condensate formation in globular proteins remain elusive. Here, we employ holographic particle characterization, a label-free, high-throughput imaging technique, to investigate the self-assembly of bovine serum albumin (BSA), a model globular protein. We show that this technique reliably differentiates between liquid-like and gel-like condensates by their distinct refractive indices and morphologies. Coupled with size-exclusion chromatography, our analysis reveals that BSA phase separation strictly depends on higher-order oligomeric assemblies. Monomeric and dimeric fractions failed to form condensates under identical crowding conditions. Furthermore, the internal packing density of these condensates is tunable via pH-driven protonation changes but remains insensitive to ionic screening. These findings support a model of "emergent multivalency," in which oligomerization increases the effective interaction valence and creates a structural scaffold that enables hydrophobically stabilized phase separation, consistent with phase separation coupled to percolation frameworks.