PORE-SIZE CONTROLLED AND POLY(ϵ-LYSINE)-IMMOBILIZED CELLULOSE SPHERICAL PARTICLES FOR REMOVAL OF LIPOPOLYSACCHARIDES

Abstract

Poly(ϵ-lysine) was covalently immobilized onto cellulose spherical particles and used for selective adsorption of pyrogenic lipopolysaccharides (LPS) from protein solutions. The resulting poly(ϵ-lysine)-immobilized cellulose particles (PL-cellulose), which had diameters of 44 to 105 μm and matrix's pore-sizes of 2 × 10³, 1 × 10⁴, and >2 × 10⁶ as molecular mass exclusions (M_lim), were used as adsorbents. The adsorption of LPS and protein to the adsorbent were determined using a batchwise method. The larger the pore size (M_lim) of the adsorbent, the larger is the LPS-adsorbing activity of the adsorbent.

The apparent dissociation constant between the LPS (E. coli O111:B4) and the adsorbent decreased from 3.8 × 10⁻¹⁰ to 1.1 × 10⁻¹¹ M with an increase in the M_lim from 2 × 10³ to >2 × 10⁶ at an ionic strength of μ = 0.05 and a pH of 7.0. On the other hand, the adsorbing activity of bovine serum albumin also increased with the increasing M_lim of the adsorbent, but sharply decreased with increasing ionic strength of the buffer.

When PL-cellulose-10³, having a small M_lim of 2 × 10³, was used as the adsorbent at a wide ionic strength of μ = 0.05 to 0.4 and a pH of 7.0, it selectively reduced LPS in various protein solutions, including 1 mg mL⁻¹ of protein and 1500 to 32,000 pg mL⁻¹ of natural LPS. When PL-cellulose-10⁶, having a large M_lim of over 2 × 10⁶, was used as the adsorbent, it only selectively removed LPS at a high ionic strength of μ = 0.4 and a pH of 7.0. The LPS-removing activity of PL-cellulose-10⁶ was always stronger than that of PL-cellulose-10³. PL-cellulose-10⁶ decreased the concentration of LPS in each protein solution to less than 10 pg mL⁻¹. On the other hand, the recovery rate (99%) of protein for PL-cellulose-10³ was higher than that for PL-cellulose-10⁶ (96 to 97%) in all cases.

ACKNOWLEDGMENTS

We are grateful to Chisso Co., Ltd. for providing poly(ϵ-lysine) and various cellulose particles. This work was supported in part by a Grant-in Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan.