Graduate Thesis Or Dissertation
 

Studies on the interaction of polylysine with deoxyribonucleic acid

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  • The interaction of polylysine with DNA may be divided into two separate phenomena: the primary interaction involved in formation of a well-defined complex, and secondary interactions leading to aggregation of these complexes. Most studies on the interaction of polylysine with DNA have used techniques which measure properties of the aggregated phase resulting from complex formation, and the properties of the interaction have been inferred from these measurements. When polylysine complexes with DNA its rotational freedom is decreased and consequently the fluorescence depolarization of a chromophore attached to the polylysine also decreases. The calculation of the fraction of polylysine bound from fluorescence depolarization measurements were found to be independent of the degree of aggregation of the complex, and independent of factors affecting aggregation. This demonstrates that it is, therefore, possible to measure the primary interaction between polylysine and DNA directly by fluorescence depolarization. In contrast, sedimentation and turbidity measurements more closely relate to the state of aggregation of the complex rather than the formation of the complex itself. The interaction has been reported in the literature to be irreversible at low ionic strength, i.e. 0.0 M to about 0.4-0.8 M NaCl, and reversible at high ionic strength, 1.0 M NaCl. This study shows that the fraction bound, of polylysine, at 1.0 M NaCl is a function of the path taken to reach 1.0 M NaCl. It is also shown that, when the DNA concentration in the sample is raised and complexes formed, the binding decreases even though, on an equilibrium model for the interaction, more binding sites would become available. Both of these findings demonstrate that the interaction between polylysine and DNA is irreversible. pH studies indicate pK shifts of the chromophore occur as the complex is dissociated by NaCl. This is interpreted as charge effects of the DNA phosphates. Neutralization of the phosphates by salt ions changes the pK of the chromophore. The turbidity is found to be sensitive to many experimental parameters and is not a quantitative measure of the complex. It is also shown that rhodamine B conjugates, as well as dansyl conjugates, are useful for studies of dye-labelled polylysine-DNA interactions.
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