Monomolecular condensation of λ‐DNA induced by cobalt hexammine

Abstract

AbstractMeasurements of static and dynamic light scattering have been used to distinguish between monomolecular DNA condensation and aggregation of condensed molecules. In low salt, using Co3+(NH3)6 as the condensing agent, and at λ‐DNA concentrations below 0.2 μg/mL, the transition curves for monomolecular condensation and aggregation are well separated for times of 16 h. In these conditions, the intensity of scattered light (90°) and also the diffusion coefficient of the condensed DNA show reasonable values for monomolecular condensation that are independent of DNA concentration and also of Na+ Co3+(NH3)6 concentrations for which monomolecular condensation is complete. At higher Co3+(NH3)6 concentrations, which produce aggregation (as judged by the intensity of scattered light), the diffusion coefficient decreases sharply.The transition curve for monomolecular condensation is independent of DNA concentration but shows a hysteresis loop. The kinetics of condensation are slow in the forward direction and fast in the reverse direction, indicating that the actual transition curve is measured closely by reversal experiments. Aggregation is blocked kinetically in both the forward and reverse directions when Co3+(NH3)6 is the condensing agent at low Na+ concentrations. When spermine or spermidine is the condensing agent and observations are made at 16 h, it is not possible to separate the transition curves for monomolecular condensation and for aggregation in conditions that are successful with Co3+(NH3)6.Some interesting properties of monomolecular condensation are noted. (1) The transition is not a two‐state reaction, as judged by measurements of the diffusion coefficient through the transition zone. (2) The transition for monomolecular condensation is diffuse. (3) The dimensions of the monomolecular condensates have been calculated from the translational diffusion coefficient for an assumed toroidal shape by the formula derived by Allison and coworkers [(1981) Biopolymers 20, 469–488]. These dimensions are in reasonable agreement with ones deduced from electron microscopy by Chattoraj and coworkers [(1978) J. Mol. Biol. 121, 327–337]. (4) The phase diagram relating the Na+ to the Co3+(NH3)6 concentrations needed for condensation has a slope of 0.6 in a log–log plot. According to numerical solutions of Manning's theory for the atmospheric binding of competing cations to DNA, this means that condensation occurs at a late stage in the replacement of Na+ by Co3+(NH3)6 around the DNA. The fraction of DNA phosphate charge neutralized at condensation is computed to be in the neighborhood of 0.9, as found by Wilson and Bloomfield [(1979) Biochemistry 18, 2192–2196], but to vary with the Na+ concentration.