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Application of SPR to Bacteriology: Endotoxin/Protein Interaction Studies
Endotoxin (commonly referred to as lipopolysaccharide in bacteriology) is associated with the outermembrane of Gram-negative bacterial pathogens such as Escherichia coli, Salmonella, Shigella, andPseudomonas [1-2]. The interaction between endotoxin and bacterial cell surface is schematically
depicted in Figure 1. Endotoxin elicits a series of pleiotropic effects on cells or organisms and istherefore harmful to most mammals. The most serious consequence of Gram-negative bacterial
infection is caused by the circulation of endotoxin and is termed as ‘endotoxic shock’ [2]. An endotoxin
molecule is less potent when it is membrane-bound. Thus pathological effects arise when endotoxin
molecules are released from multiplying cells. It is known that endotoxin and its complexes with certain
proteins in plasma can interact with the CD-14 protein, which is a type of receptors on monocytes,
macrophages and endothelial cells. This application note describes a novel application of SPR to thedetection of endotoxin via its interaction with the CD-14 protein.

EndotoxinPhospholipidBilayer

FIG. 1 The interaction of endotoxin withphospholipid bilayer of the outermembrane of Gram-negative bacterium.
The yellow circles represent the lipid
head groups and red ovals are the coresof endotoxin, and the branches are
antigens. Endotoxin molecules areembedded in the bilayer via insertion of
their lipid A chains into the membrane.
For clarity, endotoxin and phospholipid
molecules are not drawn to scale.

Before the experiment, a Au sensor chip was coated with a carboxymethylated dextran film. The
formation of the carboxymethylated dextran is straightforward and follows a protocol described inliterature [3]. Pyrogen-free water was used as the carrier (running) solution. Upon activation of thecarboxyl groups on the carboxymethylated dextran with NHS/EDC, CD14 protein solution was injected
into the SPR flow cell. CD14 can be effectively attached to the activated dextran film. Upon deactivationof the resultant surface with 1 M ethanolamine, solutions containing different amounts of endotoxin
were injected and the binding curves were recorded. Figure 2 is an overlay of a series of binding
curves.
Using a molecular weight of 10,000 Daltons for endotoxin, a KD value of 37 nM was deduced fromthe simulation and suggests that endotoxin binds strongly to CD-14 protein. Furthermore, theassociation (ka) and dissociation (kd) constants were determined to be 8.0 × 105 M-1 s-1 and 0.029 s-1,respectively. Thomas and Surolia measured the kinetics of the interaction between endotoxin andpolymyxin B (a cationic decapeptide) and determined the KD value to be about 157 nM [4].
Comparable ka and kd values were also reported [4]. Thus, endotoxin appears to bind to CD14 almost

as strongly as the peptide antibiotics. Moreover, an endotoxin concentration as low as 5 ng/mL can beeasily detected. From this note, it is evident that SPR is a suitable technique for monitoring binding ofendotoxins to proteins, antibiotics, and other species.

FIG. 2 Binding curves between pre-immobilized
CD-14 protein and different concentrations ofendotoxin: 20 ng/mL (red), 10 ng/mL (blue) and5 ng/mL (black). A flow rate of 50 µL/min wasused.

Acknowledgement: We thank Jim Powers for his technical help, discussion, and the provision of theCD-14 protein and endotoxin samples.
References[1]. Y.L. Tzeng, A. Datta, V.K. Kolli, R.W. Carlson and D.S. Stephens, J. Bacteriol. 184, 2379-88(2002).
[2]. C.R.H. Raetz, Annu. Rev. Biochem. 59, 129-170(1990).
[3]. X. Yao, X. Li, F. Toledo, C. Zurita-Lopez, M. Gutova, J. Momand and F. Zhou, Anal. Biochem. 354, 220228(2006).
[4]. C.J. Thomas and A. Surolia FEBS Lett. 445, 420-424(1999).

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