Circular dichroism investigation of the effect of plasmid DNA structure on retention in histidine chromatography

The effect of temperature-induced changes in secondary and tertiary structures of plasmid DNA (pDNA) and on the retention behaviour of open circular (oc) and supercoiled (sc) isoforms in histidine–agarose chromatography was investigated by Circular dichroism (CD) spectroscopy. Chromatographic experiments performed with three plasmids (2.7, 6.1 and 7.4 kbp) and with a decreasing ammonium sulphate gradient (2.3--2.0 M) showed that the retention of sc pDNA increased as temperature decreased from 24 to 5 °C. Such behaviour was attributed to the temperature-induced removal of negative superhelical turns in sc pDNA which is accompanied by a decrease in the number of dissociated base pairs responsible for interaction with the histidine ligands. CD spectroscopy showed that temperature has an important effect on plasmid secondary structure if adenine-rich inserts are present in the plasmid structure. Chromatographic experiments also suggested that base composition could also be responsible for the induction of specific interactions with histidine ligands.