SynopsisBinding constants and binding site sizes for the interactions of the polyamines spermine (+4), spermidine (+3), and putrescine (+2) with helical DNA have been determined as a function of ionic conditions and temperature by equilibrium dialysis using 14C-labeled polyamines. In addition, competition equilibrium dialysis has been used to determine binding parameters for the divalent cations putrescine and Mg2+ from the competitive effect of these ions on the binding of spermine or spermidine. In all cases, the logarithm of the binding constant
The quadruplex structure of the oligomer d(T2G4T) is more stable in the presence of K+ than in the presence of Na+. This enhanced stability correlates with the preferential binding of K+ to a small number of specific sites on the quadruplex. In contrast, Na+ and K+ compete on an equal footing for atmospheric binding. Both 39K+ and 23Na+ are, when specifically bound, significantly inhibited in their rotational mobility, so that the quadrupolar relaxation reflects the molecular tumbling of the oligomer, which occurs on the time scale of nanoseconds. This rotational immobilization is in distinct contrast to the high rotational mobility of atmospherically bound cations. On the other hand, all NMR-visible 39K+ in solution is in rapid exchange among all environments (free, specifically bound, and atmospherically bound) implying that the lifetime of specifically coordinated 39K+ must be significantly shorter than a millisecond. A similar conclusion holds for 23Na+. The oligomer d(T2G4T) forms two distinct Hoogsteen base-paired structures in NaCl solution, separated by a large kinetic barrier. Neither of these structures is as stable with respect to base pair opening as is the quadruplex structure formed in KCl solution. Only one of these two structures is associated with rotational immobilization of bound 23Na+.
The favorable bile acid binding characteristics of sevelamer provide a compelling explanation for its ability to lower LDL cholesterol in hemodialysis patients and in healthy volunteers.
Circular dichroism (CD) spectra of d(CCCCGGGG) in the presence of Co(NH3)6(3+) are very similar to spectra of r(CCCCGGGG). In contrast, B-form characteristics are observed for d(CCCCGGGG) in the presence of Na+ and Mg2+, even at high salt concentrations. Spermidine induces modest changes of the CD of d(CCCCGGGG). The NMR chemical shifts of the nonexchangeable protons of d(CCCCGGGG) in the absence and presence of Co(NH3)6(3+) were assigned by proton two-dimensional (2D) NOESY and COSY measurements. The chemical shifts of the GH8 protons of d(CCCCGGGG) move upfield upon titration with Co(NH3)6Cl3. The sums of the sugar H1' coupling constants decrease with added Co(NH3)6Cl3. Cross peak intensities in the 2D proton NOESY spectra show a transformation from B-DNA to A-DNA characteristics upon the addition of Co(NH3)6Cl3. The temperature-dependent 59Co transverse and longitudinal relaxation rates demonstrate that Co(NH3)6(3+) is site-bound to the oligomer. Such localization is not a general feature of Co(NH3)6(3+) binding to oligonucleotides. 59Co NMR relaxation and CD measurements demonstrate chiral discrimination by d(CCCCGGGG) for the two stereoisomers of Co(en)3(3+). Both stereoisomers bind tightly as judged by 59Co NMR, and both cause large (but nonequivalent) changes in the CD of this oligomer.
Previously, we examined the imino proton relaxation of d(GGAATTCC) in order to characterize salt and polyamine effects on the base-pair opening kinetics of this oligonucleotide [Braunlin, W. H., & Bloomfield, V. A. (1988) Biochemistry 27, 1184-1191]. Here, we report salt-dependent measurements of the NMR behavior of the nonexchangeable base proton resonances of d(GGAATTCC). From chemical shift measurements, we find an unexpectedly large salt dependence of Ka, the equilibrium constant for helix association. A total of 1.8 +/- 0.3 sodium ions are thermodynamically released upon dissociation of the octamer duplex. Most of the salt dependence of the equilibrium constant can be traced to a large salt dependence of the association rate. Thus, 1.4 +/- 0.2 sodium ions associate during the rate-limiting step of helix association. In agreement with our previous imino proton results, we also find a significant salt dependence of the duplex dissociation rate. Activation energies for helix association are very small, and possibly negative; most of the temperature dependence of the association equilibrium can be traced to a large activation energy (approximately 50 kcal/mol) for duplex dissociation.
Tolevamer, (GT160-246), is a sodium salt of styrene sulfonate polymer that is under development for the treatment of diarrhea caused by infection with Clostridium difficile. Pulsed ultrafiltration binding experiments in phosphate buffer containing 0.15 M Na(+) provide per polymer chain dissociation constants of 133 nM and 8.7 microM for the binding of tolevamer to C. difficile toxins A and B, respectively. At 0.05 M Na(+), the binding of toxin A to tolevamer is irreversible, whereas the dissociation constant to toxin B under these conditions is 120 nM. Binding constants obtained from fluorescence polarization data for toxin A binding to tolevamer at 0.15 M Na(+) agree substantially with those obtained by pulsed ultrafiltration. The binding activity of tolevamer reported here correlates well with previously reported results for the inhibition of the biological activity of C. difficile toxins A and B. From the fluorescence polarization data, it is estimated that one toxin A molecule interacts with between 600 to 1000 monomer units on tolevamer at 0.15 M Na(+). Thus, the data suggest a very large interaction surface between polymer and toxin A.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.