Holographic fingerprinting reveals oligomer-driven phase separation in Bovine Serum Albumin.

Biomolecular condensates formed through phase separation are fundamental to cellular organization. Although the physical principles underlying 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 amorphous aggregates from liquid-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 fail to form condensates under identical crowding conditions. Furthermore, the internal packing density of these condensates is highly tunable via pH-driven protonation changes but remains insensitive to ionic screening. These findings support a model of "emergent multivalency," where oligomerization creates a structural scaffold enabling hydrophobically stabilized phase separation, thereby defining a molecular threshold for globular proteins condensation.