PARTIAL CHARACTERIZATION OF Limulus polyphemus ENDOTOXIN BINDING PROTEINS, USING BIOTINYLATED LIPOPOLYSACCHARIDES

Abstract

Endotoxin, the lipopolysaccharide (LPS) component of the cell wall of gram-negative bacteria, is a constant concern to the pharmaceutical and medical device industry, due to its pyrogenic properties and ability to induce septic shock. Methods for detection and removal are important. In this project, five proteins were isolated from amebocyte lysate of the horseshoe crab Limulus polyphemus, using an endotoxin-agarose affinity column, and evaluated for specific interaction with endotoxin. These proteins were identified as Li/nu/us endotoxin-binding protein-protease inhibitor (LEBP-PI), 18K Li17111111S agglutination-aggregation factor (18K-LAF), L10, hemocyanin and a2-macroglobulin (Coneicão, et al. 1991; Nobutaka, et al. 1992; Okino, et al 1995; Lamy et al. 1983;Yokota, E. and A. Riggs, 1984; Armstrong, et al. 1990). To assess specificity, each of these proteins was evaluated for their ability to bind LPS, using biotin labeled LPS from six different gram-negative bacterial sources. Only LEBP-PI, 18K-LAF, and L10 exhibited consistent specific binding activity. In addition, the specific binding of the these proteins indicates lipid A dependence. Hemocyanin and a2-macroglobulin binding to the column matrix appeared to be the result of a nonspecific interaction or possible interaction with other basic proteins. With strong consistent lipid A specific binding properties LEBP-PI, 18K-LAF, and L10 are good candidates for potential methods of detection and/or removal of endotoxin in aqueous environments.