Absorption spectroscopy and dynamic and static light-scattering studies of ethidium bromide binding to calf thymus DNA: implications for outside-binding and intercalation

Abstract

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, T8D, is dependent on the counterion concentration, Cs T 8 D decreases slightly as C, 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 ( I ) '8D, the fraction of intercalated dye molecules per nucleotide, increases continually with T8D, (2) over the experimental accessible range of T8D, intercalation is the stronger mode of binding, and (3) dye binding appears to be saturated when '8D = T8D. 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, Dz*o. 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, I,, 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 (q2), is well simulated by the Rouse-Zimm polymer model comprised of a number of subchains and an apparent subchain d8usion coefficient DPIat that scales as TIT. In particular, it is found that the Rouse-Zimm subchain extension, RZbs, is twice the effective persistence length, 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 I, 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.

Electrochemical Studies of the Intercalation of Ethidium Bromide to DNA

“…The second mode of side binding is thought to be an association between the dye molecules and the phosphate groups at the DNA surface. The second mode is most evident at low salt and high dye concentration [18]. In this experiment, as the EB to DNA concentration ratio is low and the salt concentration is high, the second mode of binding should thus be excluded.…”

“…At room temperature, 2 hours of stirring is needed for a complete dissolution of ctDNA. The concentrations of DNA were determined spectrophotometrically by using an extinction coef®cient of 6600 M À1 at l 260 nm, based on nucleotide phosphate [18]. To prepare the EB-DNA sample solutions, an appropriate amount of EB stock solution was added to an approximate 5.0 mL of DNA stock solution and diluted to a ®nal volume of 10.0 mL.…”

Studies of Polyelectrolyte Solutions III. Correlations between the Second and the Third Virial Coefficient

“…Figure 5 contains also some experimental data obtained for Calf-Thymus DNA of M w = 6.0 X 10 6 Da. Interestingly, at small ct 3 the DNA data 34 follow those of PSS, and at large ct 3 they seem to converge to curve (1) or (2). This suggests that it may be possible to describe the exlcuded volume effects appertaining to different types of polyelectrolytes by one and the same universal interpenetration function, 'P.…”

“…Note, similar curves are obtained for DNA, PAA, and other polyelectrolytes. 34 Two fit-parameters remain in eq 34 to 38, namely X and q. The lattice-coordination number, q, is believed to be a constant over the whole range of 0(.…”

Atomic Force Microscopic Examination of Chromosomes Treated with Trypsin or Ethidium Bromide

“…6, 7) EB treatment revealed that EB could interdict cell differentiation by interfering with chromosome condensation. And virtually, EB is a cationic dye that can inhibit DNA synthesis by intercalating into the base pairs of DNA [17][18][19] and change its physical properties such as rigidity, elasticity, space structure etc., 10,11,[20][21][22] and even result in DNA molecules broken (this is approved by our other experiment about DNA damage induced by EB), then chromosomal structure damnifyed. The results indicate chromosome damage mainly locate at centromeric region (Fig.…”