Biophysical characterization of lipopolysaccharide and lipid a inactivation by lactoferrin

The interaction of bacterial endotoxins (LPS Re and lipid A, the 'endotoxic principle' of LPS) with the endogenous antibiotic lactoferrin (LF) was inv estigated using various physical techniques and biological assays. By apply ing Fourier-transform infrared (FTIR) spectroscopy, we find that LF binds t o the phosphate group within the lipid A part and induces a rigidification of the acyl chains of LPS. The secondary structure of the protein - as moni tored by the amide I band - is, however, not changed. Concomitant with the IR data, scanning calorimetric data indicate a sharpening of the acyl chain phase transition. From titration calorimetric and zeta potential data, sat uration of LF binding to LIPS was found to lie at a [LF]:[LPS] ratio of 1:3 to 1:5 m from the former and 1:10 m from the latter technique. X-ray scatt ering data indicate a change of the lipid A aggregate structure from invert ed cubic to multilamellar, and with fluorescence (FRET) spectroscopy, LF is shown to intercalate by itself into phospholipid liposomes and may also bl ock the lipopolysaccharide-binding protein (LBP)-induced intercalation of L PS. The LPS-induced cytokine production of human mononuclear cells exhibits a decrease due to LF binding, whereas the coagulation of amebocyte lysate in the Limulus test exhibited concentration-dependent changes. Based on the se results, a model for the mechanisms of endotoxin inactivation by LF is p roposed.