I have gone through some literature in which UV absorption titration for DNA binding studies has been carried out using constant DNA concentration. While some of the papers, depict the UV spectral titration of DNA and compound , by keeping compound's or drug'd concentration constant. Which method is considered more reliable? Does they have same significance towards binding mode as intercalation/ groove? Please researchers, It will be helpful for me.
The choice of what is kept constant in the titration mainly depends on the source of your spectroscopic signal (e.g. if the drug is the active species that absorbs or emits, then you want to keep its concentration constant but if, for example, you are using induced CD signal, is best to keep the DNA constant). when you add DNA over the compound its called reverse titration, and if you use calf-thymus DNA their mathematical treatment is not the same as with direct titrations. You can also add ITC, which allows you to go both ways.
A couple of good references:
W. Bujalowski and M. J. Jezewska, J. Mol. Struct., 2014, 1077, 40–50.
S. C. Kowalczykowski, L. S. Paul, N. Lonberg, J. W. Newport, J. A. McSwiggen and P. H. von Hippel, Biochemistry, 1986, 25, 1226–1240.
Dear Dr. Poonam
I agree with Vazquez answer, it is very appropriate to increase the DNA concentration for a fixed drug concentration in absorption and emission spectrophotometry. for ethidium bromide and CD method it is good to keep DNA concentration fixed and increase the concentration of drug in titration.
up vote if any useful.
for other DNA/HSA binding protocols have a look on the pdf.
But if you fixed the DNA concentration on the UV-Vis experiment and increased the complex concentration its possible to calculate the binding constant (Kb)? If so how?
M. Eugenio Vazquez
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