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Iron sulfate inhibits L imulus activity by induction of structural and qualitative changes in lipid A
Author(s) -
Fujita Y.,
Nabetani T.
Publication year - 2014
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/jam.12349
Subject(s) - limulus amebocyte lysate , lipid a , chemistry , limulus , lipopolysaccharide , coagulation , biochemistry , sulfate , sulfation , organic chemistry , biology , immunology , psychology , paleontology , psychiatry
Abstract 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.