Pore formation by whole diphtheria toxin and isolated translocation domain

We have developed a method which uses Cascade blue labeled dextrans trapped inside large unilamellar vesicles to measure pore formation by diphtheria toxin. Pore size is assayed by measuring the extent of the release of four different sized dextran molecules. Release is assayed by measuring the amount of quenching of the Cascade blue fluorescence by anti-Cascade blue antibodies post toxin addition. Using this method we determined the effect of time, protein concentration, and lipid composition on pore formation by whole diphtheria toxin and the isolated translocation domain of the toxin. Pore formation by whole toxin is rapid and complete within minutes. The size of the dextrans released from PG/PC LUVs was dependent on protein concentration. When the concentration of protein was low only the smallest molecule was released. As the toxin concentration was increased we saw the release of the 3 kD dextran followed at higher toxin concentrations by the 10 kD dextran. The 70 kD dextran was not released under our experimental conditions. These results suggest that diphtheria toxin forms oligomers with a larger pore size at high protein concentrations. We have measured the oligomerization of whole toxin using rhodamine quenching under various conditions. We found that oligomerization was pH dependent. Oligomerization was also a function of protein concentration. When toxin concentrations were low we saw small amounts of quenching suggesting the formation of small oligomers. When toxin concentration was increased, rhodamine quenching increased, suggesting the formation of larger oligomers. Pore formation by the isolated translocation domain of diphtheria toxin was protein concentration dependent and required higher amounts of protein to stimulate dextran release from LUVs than whole toxin.