Ca2+ binding to bovine lactoferrin enhances protein stability and influences the release of bacterial lipopolysaccharide.
Bovine lactoferrin (bLf) is known to damage the outer membrane of Gram-negative bacteria by binding to bacterial lipopolysaccharide (LPS). We report that LPS is released from bacterial outer membranes also when apo- or metal-saturated Lf is separated from bacterial cells by a dialysis membrane. This process occurs in phosphate-buffered saline with no added Ca2+ and Mg2+ and is hindered by addition of these cations. The effect of bLf is similar to that induced by EDTA and has been ascribed to chelation of Ca2+. In fact, it may be envisaged that Ca2+-binding sites on LPS have different affinities and that bLf can remove those ions that are more weakly bound. Ca2+ binding does not alter Lf iron-binding properties significantly or its UV and CD spectral features but brings about changes in the FT-IR bands due to carboxylate residues. Ca2+ binding is characterized by an apparent dissociation constant of 6 microM and a stoichiometry of 1.55 Ca2+ per Lf molecule; it enhances bLf stability towards chemical and thermal denaturation. The increase in stability takes place in both the apo- and iron-saturated forms but not in the desialilated protein, indicating that the carboxylate groups of the sialic acid residues present on two of the glycan chains are involved in Ca2+ binding.
Rossi
Paola
P
Department of Pure and Applied Biology, University of L'Aquila, Italy.
Giansanti
Francesco
F
Boffi
Alberto
A
Ajello
Maria
M
Valenti
Piera
P
Chiancone
Emilia
E
Antonini
Giovanni
G
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002942:Circular Dichroism; D007781:Lactoferrin / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000378:metabolism*; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D011550:Pseudomonas aeruginosa / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature
D008070:Lipopolysaccharides; D007781:Lactoferrin; D002118:Calcium