Lipopolysaccharide (LPS) is the major surface membrane component present in almost all Gram-negative bacteria and it is essential to both the form and function of the outer membrane. Lipopolysaccharides are known to be responsible for the production of various inflammatory cytokines produced by monocytes and macrophages. The role of LPS in Gram-negative bacterial diseases and its widespread application in various cell stimulation experiments provide a conceptual framework for studies targeting the isolation, purification, and detailed characterization of chemical interactions and structure of LPS. The main problem with LPS purification protocols is the contamination of the final product with proteins and nucleic acid, which hinders further quality assurance of the studies. Although, the structure and pathophysiological functions of LPS have been extensively studied, the studies of physical properties such as aggregation of LPS in aqueous solution are limited. According to other researches there is evidence that only endotoxin in aggregated structure is biologically active [1].
During the study, E.coli bacteria were grown in a bioreactor. The experiments have been performed in two different conditions: in the first group bacterial cell lysis was achieved by ultrasound applications, and in the second group methanol-chloroform extraction was executed. Further steps were identical for both groups, which included enzymatic LPS protein purification, dialysis and lyophilization. Quantity and degree of purification was determined in both groups. Concentration of LPS was identified by spectrophotometry and Limulus amebocyte lysate (LAL) test. Aggregation of LPS molecules, like any other micellization of an amphiphilic molecule, can only be characterized under specific conditions. The aim of this study was to determine critical micelle concentration (CMC) in water and phosphate buffer at different temperatures using conductometry.
Conductometrically determined critical micelle formation concentration of LPS in water at room temperature (21°C), 37°C and 50°C. Comparative analysis of CMC between two separate methods of LPS purification at different temperatures was performed.