Iron sulfate inhibits L imulus activity by induction of structural and qualitative changes in lipid A

Aims The bacterial endotoxins test ( BET ) is a sensitive assay for measuring endotoxin levels in solution and uses the limulus amebocyte lysate ( LAL ) coagulation reaction. We sought to identify the mechanisms through which certain substances interfere with the interaction between LAL and bacterial lipopolysaccharide ( LPS ). Methods and Results Endotoxin lipid A was inactivated by the addition of iron sulfate, which acted on endotoxin directly and strongly inhibited LAL coagulation activity. Size‐exclusion, anion‐exchange and reverse‐phase liquid chromatography/mass spectrometry were used to examine changes in inactivated lipid A in terms of complex formation, negative charge status, hydrophobic interaction and structure. Furthermore, we verified the involvement of the lipid A phosphoryl group in the interference of iron sulfate with lipid A ‐factor C binding activity. Iron sulfate–inactivated lipid A was cleaved at its glycosidic bond, resulting in loss of hydrophobic interactions and disruption of lipid A complexes without alteration of negative charge status and lipid A ‐factor C interaction. Conclusions Lipid A cleavage was a direct result of interfering factors, including iron sulfate, which acted on endotoxin directly to disrupt lipid A complexes rather than interfering with LAL coagulation. Significance and Impact of the Study Our data provide new insights into the mechanisms of lipid A activity.