CHARACTERIZATION AND INACTIVATION OF VEROTOXIN-1 PRODUCED BY ESCHERICHIA-COLI O157-H7

Verotoxin 1 (VT-1) produced by Escherichia coli O157:H7 was purified to homogeneity by standard liquid chromatography and FPLC with a yield of 268-mu-g from 70 g of cells. Subunits A, A1, A2, and B were separated by SDS-PAGE with a 5-30% gradient and then transferred to PVDF membrane, stained with Coomassie Brilliant Blue R-250, and partially sequenced. The molecular weights of subunits A, A1, A2, and B were 32 000, 28 000, 3700, and 4700, respectively. N-Terminal amino acid sequencing of A, A1, and A2 revealed that the A and A1 subunits have the same N-amino acid terminus and that the A2 subunit was formed by nicking prior to amino acid 252. Subunit B was sequenced through 50 amino acids. Pretreatment of VT-1 with Ca2+ and EDTA increased verotoxicity, whereas Fe3+ and various divalent cations and lipids had no measurable effect on toxicity. VT-1 toxicity was stable for up to 60 min at 70-degrees-C, but verotoxicity was reduced to 90% at 80-degrees-C for 15 min and to 99% at 80-degrees-C for 30 min. Verotoxicity was completely neutralized at 80-degrees-C for 60 min and at 85-degrees-C for times greater than 5 min. Treatment at pH 4.5 for 16 h reduced activity by 90%. Although the biological activity of ingested VT-1 is unknown, the thermal and pH tolerance of the toxin may have important practical implications for the food industry.