Affinities of different proteins and peptides for lipopolysaccharide as determined by biosensor technology.
Biosensor technology was employed to study the specific interactions of different lipopolysaccharide (LPS)-binding proteins and peptides with LPS, using an LPS-coated surface. Two methods to immobilize biotinylated LPS to streptavidin-coated sensor chips (SA-chips) were evaluated. Biotinylated LPS in PBS or biotinylated LPS, pretreated with EDTA and sodium-desoxycholate, were injected across an SA-chip, resulting in a 'high-' and 'low- mass' LPS chip, respectively. While the 'high mass' LPS chip appeared to be unstable, the 'low mass' LPS chip resulted in reproducible binding curves for bactericidal/permeability-increasing protein (rBPI21) with a binding affinity corresponding to the literature (Kd: 3.75 nM). New Kd values were obtained for serum amyloid P component (SAP, Kd: 3.9 nM), a recently discovered new LPS-binding protein, and cationic protein 18 (CAP18, Kd: 0.58 nM). Moreover, binding affinities of bioactive BPI- and SAP-derived peptides could be determined. This study shows for the first time the applicability of biosensor technology to study interactions of proteins and peptides with LPS, using an LPS-coated sensor chip.
de Haas
C J
CJ
Department of Inflammation, Utrecht University, Utrecht, The Netherlands. c.j.c.dehaas@lab.azu.nl
Haas
P J
PJ
van Kessel
K P
KP
van Strijp
J A
JA
D000209:Acute-Phase Proteins; D000595:Amino Acid Sequence; D023181:Antimicrobial Cationic Peptides; D015374:Biosensing Techniques; D001798:Blood Proteins / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008070:Lipopolysaccharides / Q000378:metabolism*; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D000683:Serum Amyloid P-Component / Q000235:genetics / Q000378:metabolism; D019809:Streptavidin; D000099231:Lipopolysaccharide-Binding Protein
D000209:Acute-Phase Proteins; D023181:Antimicrobial Cationic Peptides; D001798:Blood Proteins; D002352:Carrier Proteins; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D010455:Peptides; D011506:Proteins; D011994:Recombinant Proteins; D000683:Serum Amyloid P-Component; D019809:Streptavidin; D000099231:Lipopolysaccharide-Binding Protein; C021623:bactericidal permeability increasing protein