Iron sulfate inhibits Limulus activity by induction of structural and qualitative changes in lipid A
Article first published online: 17 OCT 2013
© 2013 The Society for Applied Microbiology
Journal of Applied Microbiology
Volume 116, Issue 1, pages 89–99, January 2014
How to Cite
Fujita, Y. and Nabetani, T. (2014), Iron sulfate inhibits Limulus activity by induction of structural and qualitative changes in lipid A. Journal of Applied Microbiology, 116: 89–99. doi: 10.1111/jam.12349
- Issue published online: 16 DEC 2013
- Article first published online: 17 OCT 2013
- Accepted manuscript online: 25 SEP 2013 02:16AM EST
- Manuscript Accepted: 15 SEP 2013
- Manuscript Revised: 30 AUG 2013
- Manuscript Received: 13 JUN 2013
- bacterial endotoxins test;
- iron sulfate and lipid A;
- limulus amebocyte lysate;
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.
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.