Investigations into Lysozyme-Endotoxin Interactions with FT-IR Spectroscopy and X-Ray Diffraction

Abstract

Lipopolysaccharide (LPS, endotoxin) as major amphiphilic component of the outer leaflet of the outer membrane of gram-negative bacteria exerts, when released from the bacteria, a variety of biological activities in mammals. It is known that LPS bind to a variety of mammalian proteins such as albumim, lysozyme, and hemoglobin, but also to the acute phase protein LBP (lipopolysaccharide-binding protein). These proteins may strongly modify the response in the biological system. For a closer characterization of the endotoxin-protein binding, we have studied the interaction of LPS with — in a first step — lysozyme. For this, we have applied IR spectroscopy to determine the s↔α acyl chain melting transition of the endotoxins by analyzing the symmetric stretch of the methylene groups vs(CH2) and synchrotron radiation X-ray diffraction to elucidate the supramolecular aggregate structure of LPS.The results for the influence of lysozyme on the s↔α acyl chain melting transition of LPS from deep rough mutant Re are plotted in Fig. 1A showing that the phase transition temperature Tc (appr. at 30 °C) and the state of order is slightly increased in the presence of lysozyme which can be taken from the shift in the peak position of vs(CH2). Concomitantly with the increase in acyl chain order a change in the supramolecular aggregate structure takes place. In the absence of lysozyme, LPS Re adopts a cubic structure (the X-ray diffraction pattern in Fig. 1B has reflections at aQ = 14.5 nm and at 6.50 nm = aQ/√5 and 4.33 nm = aQ/√11). In the presence of lysozyme, the resulting diffraction patterns (Fig. 1C, D) clearly indicate the occurrence of a multilamellar structure (reflections in equidistant ratios).