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

The interaction of hemoglobin (Hb) with endotoxins i.e. with enterobacterial deep rough mutant lipopolysaccharide (LPS) Re and the "endotoxic principle" of LPS, lipid A] was investigated using a variety of physical techniques and with two biological assays, tumor necrosis factor (TNF)-alpha induction in human mononuclear cells and the Limulus amebocyte lysate (LAL) assay. Fourier-transform IR-spectroscopic experiments indicate nonelectrostatic binding 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 with 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 secondary structures of Hb due to LPS binding. From isothermal titration calorimetry, the molar Hb] : endotoxin] binding ratio lies between 1 : 3 and 1 : 5 molar. Synchrotron radiation X-ray diffraction measurements indicate a reorientation of the lipid A aggregates from one cubic structure to another, the final structure belonging to space group Q224. The LPS-induced TNF-alpha production of mononuclear cells is enhanced by Hb, whereas in the LAL assay an LPS concentration-dependent increase or decrease was observed. Although a detailed mechanism of action cannot be given, the enhancement of LPS bioactivity can be understood in the light of the previously presented conformational concept; Hb induces an increase in the conical shape of the lipid A moiety of LPS, higher cross-section of the hydrophobic than the hydrophilic part, and of the inclination angle of the diglucosamine backbone with respect to the direction of the acyl chains.