Effective interactions and phase behavior of protein solutions in the presence of hexamine cobalt(III) chloride

**Background:** Effective protein–protein interactions govern the phase behavior of protein solutions, including liquid–liquid phase separation (LLPS) and crystallization. Understanding these interactions is crucial for structural biology and for diseases related to protein aggregation, such as cataract, Alzheimer's, and sickle cell anemia. Reentrant condensation (RC) is a phenomenon observed in acidic globular proteins with multivalent metal salts (e.g., YCl₃, LaCl₃), where protein condensation occurs only within a specific salt concentration range. Hexamine cobalt(III) chloride (Hac) is a trivalent cation known to condense DNA and RNA via electrostatic interactions and hydrogen bonding. This study systematically examines whether Hac can induce RC in four globular proteins: β-lactoglobulin (BLG), bovine serum albumin (BSA), human serum albumin (HSA), and ovalbumin (OVA).

**Methods:** Protein stock solutions (BLG, BSA, HSA, OVA) were prepared in deionized water. Hac concentrations ranged from 1 to 50 mM, and protein concentrations from 5 to 100 mg/ml. Phase behavior was first assessed by visual inspection. Small-angle X-ray scattering (SAXS) was performed at the P12 beamline (EMBL, DESY) and with a laboratory Xeuss 2.0 instrument. Data were fitted using ellipsoidal form factors combined with interaction potentials: Screened Coulombic (SC), Two Yukawa (2Y), and Sticky Hard Sphere (SHS). The reduced second virial coefficient (B₂/B₂^HS) was calculated from SHS fits. Static light scattering (SLS) and dynamic light scattering (DLS) were conducted on BSA solutions (20 mg/ml) with Hac (0–50 mM) using an ALV/CGS-3 goniometer. Autocorrelation functions were fitted with a double exponential decay to extract diffusion coefficients.

**Key Results:** Visual inspection showed no turbidity, LLPS, or aggregation for any protein-Hac combination up to 50 mM Hac. SAXS data revealed a reentrant interaction (RI) pattern: at low salt concentrations, a correlation peak indicated repulsion; as salt increased, low-q intensity rose (indicating attraction), peaked at 12 mM for BLG and 15 mM for BSA, then decreased at higher salt. The reduced second virial coefficient for BLG reached a minimum of -1.48 at 12 mM Hac, while BSA reached 0.033 at 15 mM Hac—both above the theoretical critical value of -1.56 for LLPS. For OVA and HSA, similar but weaker trends were observed. SLS of BSA showed increasing intensity up to 12 mM Hac, then decreasing. DLS revealed two decay modes; the fast diffusion coefficient decreased from 0 to 6 mM Hac, then plateaued, indicating oligomer formation. Structure factors from SAXS confirmed increasing attraction up to the critical salt concentration, followed by weakening.

**Clinical Implications:** This study demonstrates that Hac, despite its strong condensing effect on DNA/RNA, does not induce RC or LLPS in globular proteins due to its large ionic radius (420 pm) and binding mechanism that relies on hydrogen bonds—features not well-matched to protein surfaces. The weak RI observed suggests that Hac may cause only minor clustering, insufficient for pathological aggregation. These findings are relevant for understanding metal-ion-induced protein aggregation in diseases like cataract and Alzheimer's, and for biomedical applications where cobalt ions are released from implants. The results also highlight the importance of cation size and binding specificity in modulating protein interactions, which could inform the design of therapeutic strategies to prevent or promote protein condensation.