Hexafluoroisopropanol-induced catanionic-surfactants-based coacervate extraction for analysis of lysozyme.

Abstract

A coacervate extraction method, based on hexafluoroisopropanol (HFIP)-induced catanionic surfactants and coupled with a back-extraction procedure, was developed for separation and purification of proteins, using sodium dodecyl sulfate (SDS) and dodecyltrimethyl ammonium bromide (DTAB) as representative catanionic surfactants and lysozyme as a model protein. After the coacervate extraction and back extraction, the obtained lysozyme solutions were examined in terms of quantitative analysis by capillary electrophoresis, bacteriolytic activity, and circular dichroism (CD). The effects of several parameters including back-extraction solvent, HFIP content, total surfactant concentration, and SDS/DTAB molar ratio were investigated in detail on the extraction efficiency and activity of lysozyme. Under the optimized extraction conditions (66mM KH2PO4 buffer with pH6.2 as back-extraction solvent, SDS/DTAB molar ratio = 1:1mol/mol, total surfactant concentration = 30mM, HIFP concentration = 8% v/v), the extraction recovery was 89.8% (±4.7, n = 3), limit of detection was 2.2 (±0.3, n = 3) μg mL-1, and meanwhile nearly 65% of native lysozyme activity was retained. In addition, the activity and CD assays showed that SDS/DTAB molar ratio had a significant influence on the activity and structure of lysozyme after extraction. The DTAB-rich extraction systems, in which the DTAB mole fraction was equal to or larger than70%, could keep the activity and structure of lysozyme almost in the native state. Graphical Abstract Procedure of HFIP-induced SDS/DTAB coacervate extraction and back extraction of lysozyme.