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J. Biol. Chem., Vol. 278, Issue 48, 47660-47669, November 28, 2003

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

Klaus Brandenburg, Patrick Garidel¶, Jörg Andrä, Gudrun Jürgens, Mareike Müller, Alfred Blume¶, Michel H. J. Koch||, and Jack Levin**

From the Forschungszentrum Borstel, Division of Biophysics, Parkallee 10, D-23845 Borstel, Germany, ^¶Martin-Luther-Universität, Institut für Physikalische Chemie, Mühlpforte 1, D-06108 Halle/Saale, Germany, the ^||European Molecular Biology Laboratory, Deutsches Elektronen Synchrotron, Notkestrasse 85, D-22603 Hamburg, Germany, and the ^**Department of Laboratory Medicine, University of California School of Medicine and Veterans Affairs Medical Center, San Francisco, California 94121

The interaction of purified cross-linked hemoglobin (Hb) with a pentaacylated mutant lipopolysaccharide (pLPS) and the corresponding lipid A (pLA) was studied biophysically and the effects correlated with data from biological assays, i.e. cytokine induction (tumor necrosis factor-) in human mononuclear cells and the Limulus amebocyte lysate assay. Fourier transform infrared spectroscopic and Zeta-Sizer experiments indicated an electrostatic as well as a non-electrostatic binding of Hb to the hydrophilic and to the hydrophobic moieties of the endotoxins with an increase of the inclination angle of the pLA backbone, with respect to the membrane surface, from 25° to more than 50°. Small angle synchrotron radiation x-ray diffraction measurements indicated a reorientation of the lipid A aggregates from a multilamellar into a cubic structure as a result of Hb interaction. Thus, in the absence of Hb, the molecular shape of the pentaacyl samples was cylindrical with a moderate inclination of the diglucosamine backbone, whereas, in the presence of the protein, the shape was conical, and the inclination angle was high. The cytokine-inducing capability in human mononuclear cells, negligible for the pure pentaacylated compounds, increased markedly in the presence of Hb in a concentration-dependent manner. In the Limulus assay, the pentaacylated samples were active a priori, and their activity was enhanced following binding to Hb, at least at the highest protein concentrations. The data can be understood in the light of a reaggregation of the endotoxins because of Hb binding, with the endotoxin backbones then readily accessible for serum and membrane proteins. By using fluorescence resonance energy transfer spectroscopy, an uptake of the endotoxin-Hb complex into phospholipid liposomes was observed, which provides a basis for cell activation.

Received for publication, May 7, 2003 , and in revised form, September 8, 2003.

^* This work was supported by Deutsche Forschungsgemeinschaft Grants SFB 367/B8 and Br 1070/3-2, European Union Project ANEPID, the United States Veterans Administration, and the REAC Committee of the University of California School of Medicine, San Francisco. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 49-0-4537-188-235; Fax: 49-0-4537-188-632; E-mail: kbranden{at}fz-borstel.de.

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