Semi-synthesis of polymyxin-B conjugated ovalbumin: evaluation of lipopolysaccharide binding avidity and neutralization of induced tnf-alpha synthesis.
A method is described in these investigations for the semi-synthetic production of polymyxin-B conjugated ovalbumin in the form of polymyxin-B.Sulfo-SMCC.ovalbumin (PSO). The heterobifunctional "cross-linking" agent, Sulfo-SMCC was first reacted with polymyxin-B to produce a relatively pure reactive intermediate in the form of polymyxin-B.Sulfo-SMCC. Highly purified ovalbumin was then combined with the polymyxin-B.Sulfo-SMCC reactive intermediate and contaminants removed from the final PSO end product by exhaustive microdialysis. Purity of PSO was established with by high-performance cellulose acetate electrophoresis (HPCAE), and high-performance thin layer chromatography (HPTLC) analyses. Verification of polymyxin-B.Sulfo-SMCC.ovalbumin binding avidity for lipopolysaccharide (LPS) was determined by DotBLot analysis applying fluorescein isothiocyanate labeled E. coli (055:B5) LPS fractions (FITC-LPS). Efficacy of PSO to inhibit in vitro LPS-induced synthesis of tumor necrosis factor-alpha (TNF-alpha) was assessed with a tissue culture based biological assay system. In this context, semi-synthetic conjugates of PSO (0.349 microgram/ml) effectively inhibited Salmonella minnesota (RS) LPS (2.5 ng/ml well) induced TNF-alpha synthesis and corresponding cytoprotection (100%) to WEHI 164 clone 13 cell populations.
Coyne
C P
CP
Veterinary Pharmacology Research Laboratory, College of Veterinary Medicine, Mississippi State University 39762, USA.
Moritz
J T
JT
Langston
V C
VC
D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D002855:Chromatography, Thin Layer; D003432:Cross-Linking Reagents; D003720:Densitometry; D003864:Depression, Chemical; D004588:Electrophoresis, Cellulose Acetate; D004926:Escherichia coli; D008070:Lipopolysaccharides / Q000378:metabolism*; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D008301:Maleimides; D051379:Mice; D017551:Microdialysis; D010047:Ovalbumin / Q000737:chemistry*; D011112:Polymyxin B / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D012475:Salmonella; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*
D000900:Anti-Bacterial Agents; D003432:Cross-Linking Reagents; D008070:Lipopolysaccharides; D008301:Maleimides; D014409:Tumor Necrosis Factor-alpha; C071675:sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate; D010047:Ovalbumin; D011112:Polymyxin B