In vitro interaction between calf thymus DNA and Escherichia coli LPS in the presence of divalent cation Ca2+
✍ Scribed by Subrata Panja; Bimal Jana; Pulakesh Aich; Tarakdas Basu
- Publisher
- Wiley (John Wiley & Sons)
- Year
- 2008
- Tongue
- English
- Weight
- 228 KB
- Volume
- 89
- Category
- Article
- ISSN
- 0006-3525
No coin nor oath required. For personal study only.
✦ Synopsis
Abstract
With increasing addition of Escherichia coli LPS to calf thymus DNA, both dissolved in CaCl~2~, absorption maxima of DNA at 260 nm decreased gradually with the appearance of isosbastic points at both ends of spectra, which implied some binding between DNA and LPS. Hill plot of absorbance data showed that the binding interaction was positive cooperative in nature. For any fixed concentration of DNA and LPS, extent of interaction increased as concentration of CaCl~2~ was raised from 1.0 to 100 mM, signifying the electrostatic nature of the interaction, mediated through Ca^2+^ ion. Stepwise addition of EDTA, a chelating agent for divalent cations, to DNA–LPS bound complex gradually reversed the spectral shift with increase in absorbance at 260 nm, which implied opening up of the complex, that is, reversible nature of the interaction. Circular dichroism spectral changes of DNA by the addition of LPS indicated partial transition of DNA from B to A form. Isothermal titration calorimetric (ITC) study showed that the DNA–LPS binding was an exothermic and enthalpy‐driven phenomenon. Moreover, in the presence of 100 mM CaCl~2~, binding constant of the interaction was found to be 2.6 × 10^4^ M^−1^ and 3.1 × 10^4^ M^−1^ from the analysis of Hill plot and ITC result, respectively. DNA‐melting study showed that the LPS binding had increased the melting temperature of DNA, indicating more stabilization of DNA double helix. The binding of LPS to DNA made the complex resistant to digestion with endonucleases EcoRI and DNase I. © 2008 Wiley Periodicals, Inc. Biopolymers 89: 606–613, 2008.
This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at [email protected]