Ultrastructural changes in bacterial lipopolysaccharide induced by human hemoglobin

Recently published studies showed that human hemoglobin (Hb) forms stable complexes with bacterial endotoxin (lipopolysaccharide, LPS), and that these complexes have much greater biological activity than LPS alone. LPS in LPS-Hb complexes has greater filterability, is less turbid, and has a lower sedimentation velocity compared to LPS alone, changes consistent with smaller LPS size. To further characterize the physical changes in LPS induced by Hb, we examined the ultrastructure of LPS alone and LPS-Hb complexes by negative staining. We studied two rough (Re) LPS and one smooth LPS, before and after incubation with purified human HbA0. Salmonella minnesota (Re) 595 LPS was visibly turbid, and was composed mostly of ribbon-like and mesh-like structures. In addition, this LPS contained large irregular membrane-like masses, and a few smaller discoidal particles. After incubation with Hb, none of the large mesh-like, ribbon-like or membrane-like structures remained; the resultant LPS particles were discoidal particles of 5-20 nm. Escherichia coli (Re) LPS alone was comprised predominantly of ribbon-like structures; after incubation with Hb, this LPS was comprised of circular membrane-like sheets (predominantly 40-200 nm). Smooth E. coli 055:B5 LPS alone contained heterogeneous particles of 10-100 nm. Most of the largest of these particles were disintegrated by the addition of Hb. The time course of Hb-induced LPS disaggregation was studied with S. minnesota LPS because the great turbidity of this LPS allowed us to monitor its disaggregation spectroscopically. Turbidity was decreased by 50% within approximately 5 h, and was not measurable by 25 h. We propose that the toxic domains of LPS (i.e. lipid A) are cryptic in native LPS aggregates and become exposed upon binding with Hb, thereby resulting in increased biological activity of LPS-Hb complexes compared to LPS alone.