Conformational Analysis of Endotoxins from Salmonella Minnesota with Varying Sugar Chain Lengths by FT-IR Spectroscopy

Abstract

Lipopolysaccharides (LPS, endotoxins) are the major amphiphilic components of the outer leaflet of the outer membrane of Gram-negative bacteria. They are composed of an oligo/polysaccharide moiety with — depending on the bacterial mutant — varying length and a covalently-linked hydrophobic portion termed lipid A, the ‘endotoxic principle’ of LPS. It consists of a l,4′-bisphosphorylated ß-1,6-linked D-glucosamine saccharide carrying in ester- and amide-linkage up to seven hydroxylated and non-hydroxylated fatty acid residues. LPS exert a variety of biological effects in mammals. The initial event in the activation cascade of monocytes/macrophages is LPS-binding to the cell surface at specific protein ‘receptors’. The interaction with these proteins should be influenced by physicochemical properties of LPS in aqueous solutions. These properties, again, depend on ambient parameters like temperature, water content, and concentration of cations. As temperature is a constant in a given biological system, we have studied the influence of water content and cation concentration, in particular [Mg²⁺], on the conformations of various endotoxin preparations (free lipid A, rough mutant and wild-type LPS of Salmonella minnesota) applying Fourier-transform infrared (FT-IR) spectroscopy. The analysis was performed at 40 °C (i) in the water concentration range 20 to 95% w/w (without Mg²⁺) and (ii) at a water content of 80% and varying [Mg²⁺] ([LPS]:[Mg²⁺]=10:1 to 1:10 M).