Investigation into the interaction of recombinant human serum albumin with Re-lipopolysaccharide and lipid A.
The interaction of bacterial endotoxins, deep rough mutant lipopolysaccharide LPS Re and the 'endotoxic principle' lipid A, with recombinant human serum albumin (rHSA) was investigated with a variety of physical techniques and biological assays. With Fourier-transform infrared spectroscopy and differential scanning calorimetry, the influence of albumin on the acyl chain melting behavior of the endotoxins was measured. Also, the effect on the functional groups of the endotoxins, in particular with respect to their orientation, was studied, including competition experiments with polymyxin B. Furthermore, the influence of endotoxin binding to rHSA on the protein's secondary structure was investigated. The results indicate a non-electrostatic binding with no change of the backbone orientation of LPS and only a slight change of the secondary structure of rHSA. Correspondingly, the amount of charge neutralization of the endotoxins due to rHSA measured by the electrophoretic mobility exhibited only a slight reduction of the surface potential. From these measurements and isothermal titration calorimetry, the lipid:protein binding stoichiometry was estimated to [LPS]:[rHSA], 10:1 molar. The determination of the aggregate structure of the endotoxins by X-ray small-angle scattering exhibited a complex change of a cubic into a non-lamellar structure. No influence of rHSA on endotoxin intercalation into phospholipid liposomes induced by lipopolysaccharide-binding protein could be detected by fluorescence resonance energy transfer. Finally, the LPS-induced cytokine production of human mononuclear cells was only slightly increased at high molar rHSA excess, while the coagulation of amebocyte lysate in the Limulus test yielded a complex change due to rHSA binding of LPS.
Jürgens
Gudrun
G
Forschungszentrum Borstel, Biophysik, Borstel, Germany.
Müller
Mareike
M
Garidel
Patrick
P
Koch
Michel H J
MH
Nakakubo
Hiroshi
H
Blume
Alfred
A
Brandenburg
Klaus
K
D000465:Algorithms; D002152:Calorimetry, Differential Scanning; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004735:Energy Transfer; D006801:Humans; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D008027:Light; D008033:Limulus Test; D008050:Lipid A / Q000737:chemistry* / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000737:chemistry* / Q000494:pharmacology; D009000:Monocytes / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry; D012542:Scattering, Radiation; D012709:Serum Albumin / Q000737:chemistry*; D017550:Spectroscopy, Fourier Transform Infrared; D013268:Stimulation, Chemical; D013816:Thermodynamics; D014961:X-Ray Diffraction
D007202:Indicators and Reagents; D008050:Lipid A; D008070:Lipopolysaccharides; D011994:Recombinant Proteins; D012709:Serum Albumin