FIG. 2.

The cytotoxic activity of TCA-endotoxin partitions to the phenol phase in a phenol-water extraction. (A) PW-LPS (closed squares) and EP (closed circles) were prepared from S. flexneri type 1A TCA-endotoxin (open circles). These preparations were tested for cytotoxic activity at the indicated concentrations on THP-1 cells. Percent cytotoxicity in panels A, C, and E was determined by an LDH release assay. (B) Silver and colloidal-gold protein stain of S. flexneri type 1A TCA-endotoxin (lane 1), PW-LPS (lane 2), and EP (lane 3) resolved by SDS-PAGE (15% polyacrylamide). On the silver-stained gel, note the characteristic LPS ladders in both TCA-endotoxin and PW-LPS, indicative of repeating O-antigen units. The colloidal-gold protein stain illustrates that the proteins in TCA-endotoxin partition exclusively to the phenol phase (EP). (C) The cytotoxic activity in EP is stable when subjected to PK treatment but is depleted by PB-coated agarose beads. A 1-mg/ml solution of EP was either mock treated (1) or incubated with 50 μl of uncoupled agarose beads (2), 50 μl of PB-agarose beads (3), or 0.2 U of PK-agarose beads (4) for 4 h at 37°C. The beads were removed by centrifugation, and supernatants were tested for cytotoxic activity at a dilution of 1:500. (D) SDS–15% PAGE of samples shown in panel C. The gel was stained with the colloidal-gold protein stain. (E) The cytotoxic activity in S. flexneri type 1A culture supernatants is extracted by phenol. S. flexneri type 1A culture supernatants were prepared, concentrated 1,000-fold, and extracted with hot phenol. Ethanol precipitates of the phenol (closed circles) and aqueous phases (closed squares) were resuspended in equivalent volumes of PBS and tested for cytotoxicity on THP-1 cells at the indicated dilutions. As a reference, unextracted 1,000-fold-concentrated S. flexneri type 1A culture supernatant was also tested (open circles).