High-resolution capillary electrophoretic separation of supercoiled plasmid DNAs and their conformers in dilute hydroxypropylmethyl cellulose solutions containing no intercalating agent.

The three conformers of plasmid pBR322, linear, supercoiled and nicked circular forms, were separated by capillary electrophoresis (CE) in 0.1% hydroxypropylmethyl cellulose (HPMC) solution in the absence of intercalating agents and the migration order was confirmed by co-migration of enzymatically prepared corresponding DNAs. The previously observed broad peaks of supercoiled DNAs in CE are results of unresolved peaks of topoisomers which differ only in the degrees of twisting. We have demonstrated the separation of an artificial topoisomer ladder made from pBR322 and topoisomerase I. The population of topoisomers of a supercoiled DNA is dependent on sample matrices and separation conditions.

Separation of hemoglobin variants in single human erythrocytes by capillary electrophoresis with laser-induced native fluorescence detection.

Single human red blood cells, in which the hemoglobin (Hb) molecules exist in their native, tetrameric states, were analyzed. Upon injection and lysis of a cell, the tetramers were dissociated on-column into their respective polypeptide chains, separated, and detected by laser-induced native fluorescence detection with 275-nm excitation. This technique was applied to the determination of hemoglobin variants as found in adult (normal and elevated Hb A1) and fetal erythrocytes. Normal adult cells contained 9.6% and 4.8% glycated beta- and alpha-chains, respectively. Cells with elevated Hb A1 gave 30% and 12%, respectively. The amounts of glycated Hb and total Hb in a given cell were found to be uncorrelated.