Neutralization of Human Serum β-Lysin by Sodium Polyanetholsulfonate and Sodium Amylosulfate

Abstract

Normal fresh and heat-inactivated (56 degrees C, 30 min) human sera (80 vol%, i.e., 80% [vol/vol] of a 2-ml assay volume) killed Bacillus subtilis ATCC 6633 cell inocula of 1.5 x 10(4) colony-forming units per ml within 1 to 2 h after exposure. The B. subtilis assay strain proved slightly and reversibly susceptible to 5 mug of egg white lysozyme per ml. Seitz filtration of fresh human serum completely removed beta-lysin activity; significant amounts of serum lysozyme were removed as well, as determined with the bioassay strain Micrococcus lysodeikticus ATCC 4698. However, bactericidal activity of human serum via classical or alternative complement pathway activation remained intact. Addition of 0.01 M dithiothreitol to fresh human serum abolished beta-lysin activity, but not that of serum lysozyme. Chelation of fresh and heat-inactivated human serum with 0.01 M MgCl(2) + 0.01 M ethylene glycol tetraacetic acid, but not with 0.01 M ethylenediaminetetraacetic acid, markedly retarded beta-lysin activity; however, lysozyme activity remained unaffected. Chelation of serum with 0.01 M MgCl(2) + 0.01 M ethylene glycol tetraacetic acid + 0.01 M CaCl(2) completely abrogated beta-lysin activity, but not that of lysozyme. Absorption of human serum with 10 mg of bentonite per ml (10 min, 37 degrees C) completely removed beta-lysin and lysozyme activity, but failed to affect serum bactericidal activity against Escherichia coli control strain C. Reconstitution of 50 vol% of bentonite-absorbed serum with 40 vol% of heat-inactivated human serum restored both beta-lysin and lysozyme activity. Addition of either 63 to 500 mug of sodium polyanetholsulfonate per ml or 63 to 500 mug of sodium amylosulfate per ml to 80 vol% of fresh human serum completely neutralized beta-lysin activity for the entire observation period of 22 h.