lactoferrin and potassium-thiocyanate

The ability of Shigella flexneri to interact with lactoferrin (Lf) was examined with a 125I-labeled protein-binding assay. The percent binding of human lactoferrin (HLf) and bovine lactoferrin (BLf) to 45 S. flexneri strains was 19 +/- 3 and 21 +/- 3 (mean +/- standard error of the mean), respectively. 125I-labeled HLf and BLf binding to strain M90T reached an equilibrium within 2 h. Unlabeled HLf and BLf displaced the 125I-HLf-bacteria interaction in a dose-dependent manner. The Lf-bacterium complex was uncoupled by KSCN or urea, but not by NaCl. The interaction was specific, and approximately 4,800 HLf binding sites (affinity constant [Ka], 690 nM) or approximately 5,700 BLf binding sites (Ka, 104 nM) per cell were estimated in strain M90T by a Scatchard plot analysis. The native cell envelope (CE) and outer membrane (OM) did not reveal Lf-binding components in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, after being boiled, the CE and OM preparations showed three distinct horseradish peroxidase-Lf reactive bands of about 39, 22, and 16 kDa. The 39-kDa component was also reactive to a monoclonal antibody specific for porin (PoI) proteins of members of the family Enterobacteriaceae. The Lf-binding protein pattern was similar with BLf or HLf, for Crb+ and Crb- strains. The protein-Lf complex was dissociable by KSCN or urea and was stable after treatment with NaCl. Variation (loss) in the O chain of lipopolysaccharide (LPS) markedly enhanced the Lf-binding capacity in the isogenic rough strain SFL1070-15 compared with its smooth parent strain, SFL1070. These data establish that Lf binds to specific components in the bacterial OM; the heat-modifiable, anti-PoI-reactive, and LPS-associated properties suggested that the Lf-binding proteins are porins in S. flexneri.

Topics: Bacterial Adhesion; Bacterial Outer Membrane Proteins; Binding Sites; Electrophoresis, Polyacrylamide Gel; Hot Temperature; Immunoblotting; Lactoferrin; Lipopolysaccharides; Porins; Shigella flexneri; Sodium Chloride; Thiocyanates; Time Factors; Urea

Bovine lactoferrin, isolated from colostral milk, interacted strongly with immobilized Cibacron blue F3GA column. Lactoferrin, adsorbed on the dye column, could not be eluted by 8 M urea, 1% Triton X-100, and 75% ethylene glycol, but was eluted by .1 M sodium hydroxide, 1 M potassium thiocyanate, 3 M potassium chloride, and free Cibacron blue F3GA. Electrostatic forces between the sulfonic groups of Cibacron blue F3GA and the basic side-chain groups in lactoferrin molecule probably are responsible for the observed interaction. The elution profile for lactoferrin differed from those of lactoperoxidase and serum albumin, which might allow efficient isolation of lactoferrin from whey via affinity chromatography.

Topics: Adsorption; Animals; Cattle; Chromatography, Affinity; Coloring Agents; Colostrum; Hydrogen-Ion Concentration; Lactoferrin; Lactoperoxidase; Potassium Chloride; Serum Albumin, Bovine; Sodium Hydroxide; Thiocyanates; Triazines; Urea