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 7.4kbp) and with a decreasing ammonium sulphate gradient (2.3--2.0M) showed that the retention of sc pDNA increased as temperature decreased from 24 to 5 degrees 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.
Sousa
F
F
CICS-Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, 6201-001 Covilhã, Portugal.
Prazeres
D M F
DM
Queiroz
J A
JA
D002845:Chromatography / Q000379:methods*; D002942:Circular Dichroism / Q000379:methods*; D004269:DNA, Bacterial / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013696:Temperature
D004269:DNA, Bacterial; D006639:Histidine