Chaotropic agents cause disaggregation and enhanced activity of Pasteurella haemolytica leukotoxin

Pasteurella haemolytica biotype A, serotype 1 grown to late logarithmic growth phase in cell culture medium (RPMI 1640) produced highly aggregated leukotoxin. The multimer mass of the highly aggregated leukotoxin in 0.1 M sodium phosphate buffer pH 7.0 as determined by gel filtration chromatography on Sephacryl S400HR was approximately 8000 kDa. Resuspension of leukotoxin in phosphate buffer containing various chaotropic agents resulted in partial disaggregation of leukotoxin and enhanced leukotoxic activity. 3M guanidine disaggregated leukotoxin to a multimer mass of approximately 800 kDa and enhanced leukotoxic activity 3 to 20-fold. In 6 M urea or 1 M sodium thiocyanate, leukotoxin multimers were observed ranging in mass from 8000 kDa to 400 kDa, and activity enhancement was less than that for leukotoxin in 3 M guanidine. Several detergents were tested for enhancement of leukotoxic activity, but only 1% Tween 20 enhanced leukotoxic activity (4-fold), whereas 1.25% octylglucoside, 10 mM CHAPS, and 5 mM deoxycholate diminished and 1% Triton X-100 abolished leukotoxic activity.