Characterization of the binding of endogenous proteins to endotoxins: binding stochiometry and protein secondary structure

Abstract

Lipopolysaccharide (LPS, endotoxin) as major components of the outer leaflet of the outer membrane of Gram-negative bacteria exert, when released from the bacterial cells, a variety of biological effects in mammals. Various abundant endogenous LPS-binding proteins may, however, modify the response to LPS. Therefore, for an understanding of the interaction processes the characterisation of endotoxin binding to the proteins is of importance. For an determination of the binding stoichiometry, we have performed isothermal titration calorimetric (ITC) measurements of the interaction of endotoxins (deep rough mutant LPS Re and the ‘endotoxin principle’, free lipid A) with albumin and hemoglobin. The role of the former protein as binding protein is established since long, however, up to now no unequivocal data are available regarding the influence on the biological action of LPS. The latter protein, in contrast, is known to enhance the biological action of LPS.