Liquid-liquid phase transition of protein aqueous solutions isothermally induced by protein cross-linking.
We experimentally demonstrated that liquid-liquid phase separation (LLPS) of protein aqueous solutions can be induced by isothermal protein oligomerization. This phenomenon is analogous to LLPS induced by the polymerization of small organic molecules in solution. Specifically, using glutaraldehyde for protein cross-linking, we observed the formation of protein-rich liquid droplets for bovine serum albumin and chicken egg lysozyme at 25 degrees C. These droplets evolved into cross-linked protein microspheres. If the aqueous solutions of the protein monomer do not show LLPS at temperatures lower than the oligomerization temperature, protein-rich droplets are not observed. We experimentally linked the formation of these droplets to the increase of LLPS temperature during protein oligomerization. When macroscopic aggregation competes with LLPS, a rationale choice of pH, polyethylene glycol, and salt concentrations can be used to favor LLPS relative to aggregation. Although glutaraldehyde has been extensively used to cross-link protein molecules, to our knowledge, its use in homogeneous aqueous solutions to induce LLPS has not been previously described. This work contributes to the fundamental understanding of both phase transitions of protein solutions and the morphology of protein condensed phases. It also provides guidance for the development of new methods based on mild experimental conditions for the preparation of protein-based materials.
Wang
Ying
Y
Department of Chemistry, Texas Christian University, Fort Worth, Texas 76129, USA.
Annunziata
Onofrio
O
D000818:Animals; D002417:Cattle; D002645:Chickens; D004531:Eggs; D005976:Glutaral / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008863:Microspheres; D009113:Muramidase / Q000737:chemistry*; D044367:Phase Transition; D011092:Polyethylene Glycols / Q000737:chemistry; D012492:Salts / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D012996:Solutions / Q000737:chemistry; D013696:Temperature; D014867:Water / Q000737:chemistry
D012492:Salts; D012996:Solutions; D014867:Water; D012710:Serum Albumin, Bovine; D011092:Polyethylene Glycols; D009113:Muramidase; D005976:Glutaral