On the basis of the asymmetrical charge distribution of Escherichia cob DNA topoisomerase I, we developed a new procedure to purify E. cob DNA topoisomerase I in the milligram range. The new procedure includes using both cation- and anion-exchange columns, i.e., SP-Sepharose FF and Q-Sepharose FF columns. The E. coli DNA topoisomerase I purified here is free of DNase contamination. The kinetic constants of the DNA relaxation reaction of E. coli DNA topoisomerase I were also determined. (c) 2011 Elsevier Inc. All rights reserved.