Investigation into the interaction of recombinant human serum albumin with Re-lipopolysaccharide and lipid A

Abstract

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.

Cross-linked Hemoglobin Converts Endotoxically Inactive Pentaacyl Endotoxins into a Physiologically Active Conformation

“…The transitions observed in the DSC experiments indicate a very complex protein melting as compared, for example, to human serum albumin (23). The partial melting temperatures at 63.5 and 75°C and the high denaturation temperature of 85°C for ␣␣Hb ( Fig.…”

“…Zeta Potential-Zeta potentials were determined with a Zeta-Sizer 4 (Malvern Instruments, Herrsching, Germany) at a scattering angle of 90°from the electrophoretic mobility by laser-Doppler anemometry as described previously (23). The zeta potential was calculated according to the Helmholtz-Smoluchovski equation from the mobility of the aggregates in a driving electric field of 19.2 V⅐cm Ϫ1 .…”

“…The pLPS aggregates were obtained by sonication as described previously (22,23). A known amount of protein was added to the lipid dispersion at room temperature and the mixture slightly vortexed until full protein dissolution.…”

Biophysical Characterization of the Interaction of Endotoxins with Hemoglobins

“…The interphase layer of MNC was collected and washed twice in Hank's medium and then once in serum-free RPMI 1640 which contained 2 mM Lglutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. The cells were resuspended in serum-free medium and the cell number was equilibrated at 5x10 6 Immunological determination of TNFα in the cell supernatant was performed in a sandwich-ELISA as described elsewhere [15]. 96-well plates (Greiner, Solingen, Germany) were coated with a monoclonal antibody against TNF (clone 6b from Intex AG, Muttant, Switzerland).…”

Investigation into the interaction of the bacterial protease OmpT with outer membrane lipids and biological activity of OmpT:lipopolysaccharide complexes

“…LPS aggregates were obtained by sonication as described previously (Brandenburg et al 2002b;Ju¨rgens et al 2002). A known amount of protein was added to the lipid dispersion at room temperature and the mixture slightly vortexed until full protein dissolution.…”