Synthesis of polyampholytes and their phase separation with enzymes

Complex coacervation is defined as a liquid-liquid phase separation including two phases, one dilute phase called supernatant and one dense phase called coacervate. Coacervation can be used as an encapsulation method that allows for protection of active substances like enzymes from environmental factors; e.g. pH, salt concentrations and temperature. In this method, although oppositely charged polyelectrolytes are mostly used, study of coacervation involving polyampholytes, which are polyelectrolytes with both positive and negative charges, is essential regarding their mimicry of intrinsically disordered proteins, which also carry oppositely charges on their backbone. The aim of this study is to synthesize hyaluronic acid-based polyampholytes (PA) with different grafting ratios with using 1,4-Diaminobutane dihydrochloride (DN), adipic dihydrazide (ADH) and ethylenediamine dihydrochloride (EDA) via carbodiimide chemistry. To characterize these polyampholytes, 1H-Nuclear Magnetic Resonance (1H-NMR) and Fourier-Transform Infrared (FTIR) spectroscopy were used to examine the chemical structure of the modified HA. Zeta potential experiments were conducted to obtain the isoelectric points (pI) of the polyampholytes. Synthesized polyampholytes were used to encapsulate enzymes with different isoelectric points (pI) like alpha-chymotrypsin (αCT) (pI = 8.76), carbonic anhydrase (CA) (pI = 5.9) and lysozyme (LYS) (pI = 11). Turbidimetric titration experiments with changing pH and NaCl concentration were conducted to determine the range where phase separation takes place at the optimum pH of the selected enzymes.