Studies on DNA-recipient interaction. I. Investigation of the interaction between recipients and transfection stimulators with fluorescent probes.

Abstract The interaction of some basic proteins (protamine, thymus histone and the internal protein of T2H bacteriophage) with Escherichia coli spheroplasts and its membranes were investigated with the aid of two fluorescent probes (5-dimethylamino-1-naphthalenesulfonylchloride (DNS)) and 1-anilino-naphthalene-8-sulfonate (ANS). It was found that not less than 40% (and presumably more) of the total (15 μg · ml −1 ) DNS-protamine was bound to spheroplasts. Some protein was found in isolated membranes and inside the cell. The double-reciprocal plot analysis showed that the binding of protamine resulted in the reduction of the binding sites for ANS and a great enhancement of the fluorescence yields. Addition of internal protein did not affect the number of binding sites and only slightly influenced the quantum yields. It was found that protamine and internal protein alone, in contrast to histone, did not interact with ANS. The dependence of fluorescence intensity upon protein concentration exhibited saturation kinetics for protamine and internal protein in contrast to histone where no saturation was observed. The values of saturating protamine concentration agreed well with the optimal concentration of the protein in transfection assays. Protamine treatment of spheroplasts at 2°C caused their substantial aggregation. The results were interpreted in terms of the structural and charge changes of the surface of the spheroplasts. The interaction of protamine with spheroplasts was directly related to the enhancement of their transfection by various DNAs.