A new method for isolation ofeukaryotic topoisomcrase 1 from calf thymusand from Jurkat-l cells using HPLC has been developed. The method allows quantitative purificntion of high molLvular weight topo I and of two low molecular weight fractions differing by their isoelectric points. It has been suggested that these fractions bc characterized as two subforms of the enzyme possessing structural and functional diffcrcnccs. The differences in their specific activities, sensitivity to camptothccin and in their protcolytic digestion maps have been demonstrated for the two cnzymcs.Topoisomerasc I; Subform; HPLC; Hydrophobic interaction chromatography; Camptotbecin rcsistancc lNTRODUCTlONThe type I topoisomerases (topo I) are key er~qmcs in the control of DNA topological state changes neccssary for many genetic processes [l].The procedure for isolation of topo I from calf thymus [2] and some other tissues [3] yields several polypeptides of different molecular weight. The same phenomenon was found in prokaryotic cells [4]. This might be due to partial proteolysis of the native topo I of high molecular weight. Here we suggest that this phenomenon can be caused also by another reason. EXPERIMENTAL PtwiJiccttion of type I ropoisottwoscTopo 1 was initially purified from calf thymus and Jurkat-I cells as described [3]. Then we developed our original HPLC protocol. The Altex (Beckman, Model 332) HPLC system WBS used. All steps were carried out at 4OC with buffers containing 1.4 mM 2-mercaptorthanol (M)3) and I mM PMSF; the flow rate was 0.5 ml/min.The active fraction cluting from a heparin-Scpharose column was dilutLti to a concentration of 5% glycerol. Solid ammonium sulfate (AS) was slowly added to 33% saturation, and after stirring for GO min the precipitate was removed by ccnlrifugation (I 3,000 x 6, I5 min). The supernatnnt was applied to Phenyl SPW (TSK column), cquili- bratcd with buffer A, containing 50 mM potassium phosphate buffer (KPB), pH 7.3. and 33% saturation with AS. Elution was performed with an increasing linear gradient of buffer B, containing 50 mM KPB, pH 7.2. and 30% cthylcnc glycol. After dialysis against 50 mM KPB, pH 7.2, and 20% glycerol (buffer C), specific topo I activity was found in the bound and unbound fractions. The last was loaded onto a Mono Q column, and the unbound fraction from the Mono Q coiumn was then loaded directly onto a Mono S column: at this point all the activity was bound to the column. Both columns were ctquilibratcd with buffer C and in both cases the elution was performed with aa increasing linear gradient of 50 mM KPB, pH 7.2, 20% glycerol and 1 M N&l (buffer D). Other proceduresTopo I activity was measured by the relaxation ofsuperwiled pIas_ mid DNA (pUCl9) [5]. One unit was defined as the amount of cnzymc that relaxes half of the added plasmid DNA (1 pg) in IO min at 25%. The assay was also carried out with eamptothccin (CPT) at different concentrations. Isoelectric focusinfi with subseauent immunodctcc-