Kinetic aspects of the aggregation of Clostridium perfringens theta-toxin on erythrocyte membranes. A fluorescence energy transfer study.

Fluorescence resonance energy transfer was used to monitor aggregation kinetics of the "thiol-activated" cytolysin (perfringolysin O (PFO) or theta-toxin) of Clostridium perfringens on erythrocyte membranes. PFO was labeled with the isothiocyanate derivatives of either fluorescein or tetramethylrhodamine. No detectable change in the hemolytic activity of PFO was detected after modification with either fluorophore at a ratio of 1:2 fluorophore molecules/cytolysin molecule. Fluorescence energy transfer (FET) between the donor (fluorescein.PFO or PFOD) and the acceptor (tetramethylrhodamine.PFO or PFOA) was detected by both quenching of donor fluorescence (520 nm) and by enhancement of acceptor fluorescence (575 nm) upon aggregation of labeled cytolysin molecules. FET was only observed when PFOD and PFOA were incubated in the presence of membranes. FET was not observed when PFOD and PFOA were incubated in a membrane-free solution or when unlabeled toxin was substituted for PFOA. FET was also found to be temperature-dependent. The temperature-dependent rates of change in FET upon mixing labeled toxin with erythrocyte membranes proceeded without a lag phase and displayed an activation energy of 18.7 kcal/mol. At all temperatures aggregation of PFO was virtually complete before the onset of hemolysis, the latter exhibiting a distinct lag phase. The lag period before onset of hemolysis was temperature-dependent and exhibited an activation energy of 23.2 kcal/mol. These results suggest that the aggregation of membrane-associated PFO is necessary to initiate the hemolytic process, and the lag phase which occurs before onset of hemolysis reflects the kinetics of PFO monomer to polymer conversion.