Protein Self-Assembly States Modulate Lithium Carbonate Biomineralization: From Ion Chelation to Nucleation Sites

Understanding protein-salt interactions is important for controlling crystallization, including biomineralization, biopharmaceutical purification, biocatalytic enzymes, and environmental biointerfaces. This study for the first time investigated interactions between three proteins (lysozyme, red fluorescence protein, and bovine hemoglobin) and Li₂CO₃ (formation from the reaction of LiCl with Na₂CO₃) under different protein and salt concentrations. For the crystallization of Li₂CO₃, at low supersaturation (S), the proteins inhibited Li₂CO₃ nucleation by 20-40% through chelation. At high S, the proteins accelerated nucleation by 10-40%. The dual effects of the protein on Li₂CO₃ biomineralization have been discussed. With the increase S of Li₂CO₃, the dispersion state of proteins in solution undergoes a transition from dimers to oligomers and finally to aggregates. In all ranges of S, the protein reduced the agglomeration of Li₂CO₃ crystals. In lysozyme crystallization, increasing the Li₂CO₃ concentration yielded a larger number of smaller crystals. At equal concentration of lysozyme, twice more of LiCl and Na₂CO₃ in the solution led to more than 5 times the crystal number and 5 times smaller average crystal size. The interactions among protein molecules, salt ions in solution, and Li₂CO₃ crystals have been discussed. Dynamic light scattering measurements and the fluorescence microscopy image suggest that the dual effect of proteins on Li₂CO₃ crystallization at different supersaturation levels is associated with protein molecular aggregation under varying salt concentrations, resulting in both thermodynamic and kinetic influences on the crystallization process.