ABSORPTION-SPECTROSCOPY AND DYNAMIC AND STATIC LIGHT-SCATTERING-STUDIES OF ETHIDIUM-BROMIDE BINDING TO CALF THYMUS DNA - IMPLICATIONS FOR OUTSIDE BINDING AND INTERCALATION

By combining absorption and light-scattering spectroscopy, including photon correlation and angular distribution of absolute scattered intensity, we have studied the modes of ethidium bromide binding to calf thymus DNA at constant temperature and pH. It is found that the fraction of total bound dye molecules per nucleotide, (T)theta(D), is dependent on the counterion concentration, C(S). (T)theta(D) decreases slightly as C(S) is raised. Furthermore, the data indicate two modes of dye binding: intercalation and outside binding. The analysis of the Scatchard plots by Manning's polyelectrolyte theory of site-binding equilibria suggests (1) (I)theta(D), the fraction of intercalated dye molecules per nucleotide, increases continually with (T)theta(D), (2) over the experimental accessible range of (T)theta(D), intercalation is the stronger mode of binding, and (3) dye binding appears to be saturated when (I)theta(D) almost-equal-to (T)theta(D). The light-scattering measurements gave useful data for the DNA radius of gyration, [S]z, the DNA radius of hydration, Rh, the second virial coefficient, A2, and the DNA translational diffusion coefficient, D(z,0). These data were analyzed in terms of the wormlike chain and the sliding rod model. It is found that each intercalated EBr molecule adds a length of 0.27 nm to the total contour length of CT-DNA. Interestingly, the DNA persistence length of bending, l(p), is smaller in the presence than in the absence of EBr This indicates that dye binding reduces the DNA rigidity due to a neutralization of phosphate groups. Finally, it was shown that the angular dependence of the apparent diffusion coefficient, D(app)(q2), is well simulated by the Rouse-Zimm polymer model comprised of a number of subchains and an apparent subchain diffusion coefficient D(plat) that scales as T/eta. In particular, it is found that the Rouse-Zimm subchain extension, (RZ)b(S), is twice the effective persistence length, l(p,eff), which is the weighted sum of the persistence length due to bending, twisting, and contraction/extension. A further result is that the ionic strength dependence of l(p) can be well described by Manning's theory of territorial counterion binding and that a process such as spontaneous transient opening of DNA base pairs becomes important, if at all, when the ionic strength tends to zero.