In vitro interaction between calf thymus DNA and Escherichia coli LPS in the presence of divalent cation Ca2+.
With increasing addition of Escherichia coli LPS to calf thymus DNA, both dissolved in CaCl2, 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 CaCl2 was raised from 1.0 to 100 mM, signifying the electrostatic nature of the interaction, mediated through Ca2+ 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 CaCl2, binding constant of the interaction was found to be 2.6 x 10(4) M(-1) and 3.1 x 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.
Panja
Subrata
S
Department of Biochemistry and Biophysics, University of Kalyani, Kalyani 741235, West Bengal, India.
Jana
Bimal
B
Aich
Pulakesh
P
Basu
Tarakdas
T
D000818:Animals; D002118:Calcium / Q000737:chemistry*; D002412:Cations; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry*; D008070:Lipopolysaccharides / Q000737:chemistry* / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
D002412:Cations; D008070:Lipopolysaccharides; D004247:DNA; C427203:calf thymus DNA; D002118:Calcium