Thermo-Resistive Phase Behavior of Trivalent Ion-Induced Microscopic Protein-Rich Phases: Correlating with...

Proteins, in the presence of trivalent cations, exhibit intriguing phase behavior which is contrasting compared to mono- and divalent cations. At room temperature (RT), trivalent cations induce microscopic liquid-liquid phase separation (LLPS) in which a protein-rich phase coexists with a dilute phase. The critical solution temperature related phenomena in these complex fluids are well studied; however, such studies have mostly been restricted below the denaturation temperature (T_M) of the protein(s) involved. Here, we probe the phase behavior of bovine serum albumin (BSA) incubated at 70 °C (>T_M) in the presence of Na⁺, Mg²⁺, La³⁺, Y³⁺, and Ho³⁺ ions. BSA in the presence of mono- and bivalent ions forms an intense gel phase at 70 °C; however, the trivalent salts offer remarkable thermal resistivity and retain the fluid LLPS phase. We determine the microscopic phase behavior using differential interference contrast optical microscopy, which shows that the LLPS droplet structures in the M³⁺ ion-containing protein solutions prevail upon heating, whereas Mg²⁺ forms composed cross-linking gelation upon thermal incubation. We probe the interior environment of the protein aggregates by ps-resolved fluorescence anisotropy measurements using 8-anilino-1-naphthalenesulfonic acid (ANS) as an extrinsic fluorophore. It reveals that while the LLPS phase retains the rotational time constants upon heating, in the case of gelation, the immediate environment of ANS gets significantly perturbed. We investigate the explicit protein hydration at RT as well as at T > T_M using the ATR THz-FTIR (1.5-22.5 THz) spectroscopy technique and found that hydration shows strong ion specificity and correlates the phase transition behavior.