Journal
ANALYTICAL BIOCHEMISTRY
Volume 430, Issue 2, Pages 203-213Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2012.08.027
Keywords
Lipopolysaccharides; Aggregates; Gel electrophoresis; Dynamic light scattering; Zeta potential; TLR4
Funding
- Alexander von Humboldt Foundation
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Nanoaggregates composed of selected glycoforms from Escherichia coli 055:B5 lipopolysaccharide (LPS) were prepared by combining sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, zinc-imidazole reverse staining, zinc chelation after cutting gel slices, elution with either 0.5% triethylamine (TEA) or 0.4% to 0.5% surfactant (SDS or deoxycholate [DOC]) from extrusion-generated gel microparticles, and centrifugal diafiltration after appropriate surfactant dilution. Dynamic light scattering allows detecting these aggregates, giving a size distribution from 10 to 100 nm in diameter. The formation of the aggregates prepared with selected DOC-eluted LPS glycoforms was notably improved over those prepared with TEA-eluted glycoforms. As the O-side chain length increased in the composition of the former aggregates, there was a gradual decrease in the electrophoretic mobility (from -1.2 to 0.01 10(-8) m(2)/V s), giving a calculated zeta potential from -15 to 0.1 mV at pH 6.8. These aggregates were further characterized for their abilities to elicit agonistic effects on human Toll-like receptor 4, as shown by in vitro activation of nuclear factor kappa light chain enhancer of activated B cells (NF-kappa B) in engineered HEK293 cells. (C) 2012 Elsevier Inc. All rights reserved.
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