Interaction of hemoglobin with enterobacterial lipopolysaccharide and lipid A - Physicochemical characterization and biological activity

The interaction of hemoglobin (Hb) with endotoxins [i.e. with enterobacteri al deep rough mutant lipopolysaccharide (LPS) Re and the 'endotoxic princip le' of LPS, lipid A] was investigated using a variety of physical technique s and with two biological assays, tumor necrosis factor (TNF)-alpha inducti on in human mononuclear cells and the Limulus amebocyte lysate (LAL) assay. Fourier-transform IR-spectroscopic experiments indicate nonelectrostatic b inding to the hydrophobic moiety with a slight rigidification of the lipid A acyl chains, and an increase in the inclination of the lipid A backbone w ith respect to the membrane surface from 35 degrees to more than 40 degrees due to Hb binding, but no change of the predominantly alpha -helical secon dary structures of Hb due to LPS binding. From isothermal titration calorim etry, the molar [Hb] : [endotoxin] binding ratio lies between 1 : 3 and 1 : 5 molar. Synchrotron radiation X-ray diffraction measurements indicate a r eorientation of the lipid A aggregates from one cubic structure to another, the final structure belonging to space group Q(224). The LPS-induced TNF-a lpha production of mononuclear cells is enhanced by Hb, whereas in the LAL assay an LPS concentration-dependent increase or decrease was observed. Alt hough a detailed mechanism of action cannot be given, the enhancement of LP S bioactivity can be understood in the light of the previously presented co nformational concept; Hb induces an increase in the conical shape of the li pid A moiety of LPS, higher cross-section of the hydrophobic than the hydro philic part, and of the inclination angle of the diglucosamine backbone wit h respect to the direction of the acyl chains.