THE ACCUMULATION AND COMPARTMENTALIZATION OF ISOMETAMIDIUM CHLORIDE IN TRYPANOSOMA-CONGOLENSE, MONITORED BY ITS INTRINSIC FLUORESCENCE

Interaction of the trypanocide isometamidium chloride with components of Trypanosoma congolense results in characteristic shifts in the intr insic fluorescence of the drug. The specificity of this interaction wa s investigated by analysing the effects of various physicochemical man ipulations on its fluorescence properties. The characteristic shifts i nvolved a preferential increase in the intensity of one emission peak over the other, resulting in a systematic increase in the ratio of flu orescence intensities. These effects were apparently due to constraint s on fluorophore free rotation in the solution (that is, viscosity). P urified DNA produced similar effects in a saturable manner displaying high affinity for the drug, indicating that the constraint involves bi nding of the drug to high-affinity binding sites within the DNA. Such binding sites were demonstrated in lysates derived from trypanosomal c ells. The binding sites were associated with macromolecular species (M (r)> 12000), and were partly disrupted by thermal denaturation and pro teolysis. Treatment with DNase 1 produced high levels of disruption of the binding sites (> 85 %), indicating an involvement of DNA in the b inding. BSA demonstrated weak non-specific binding of the drug. Entry of drug into live trypanosomal cells (monitored by C-14-labelled drug uptake) was paralleled by fluorescence shifts observed under comparabl e conditions of drug concentration and buffer conditions. Both systems (fluorescence shifts and accumulation of labelled drug) indicated the presence of a saturable membrane transporter with high affinity for t he drug. We conclude that monitoring the fluorescence shifts of isomet amidium constitutes a sensitive and highly specific probe for entry of the drug into trypanosomal cells, thereby enabling resolution of the transport events involved.