AbstractWe previously reported that a complex of nuclear proteins from HeLa cells, among them histone H1 and casein kinase 2 co‐eluted from immobilized nucleosome assembly protein 2 (NAP‐2)‐Sepharose. Here, using HeLa cell nuclear extracts, we found NAP‐2 migrates in a blue‐native polyacrylamide gel with an apparent molecular weight of 300 kDa. HeLa cell NAP‐2, labeled in vivo with radioactive orthophosphate, co‐precipitated with at least two phosphoproteins, with an apparent mass of 100 and 175 kDa, respectively, as determined by SDS–PAGE. NAP‐2 from total HeLa cell extract co‐purified with other proteins through two sequential chromatographic steps: first, a positively charged resin, Q‐Sepharose, was used, which purified NAP‐2 more easily with other proteins that eluted as a single peak at 0.5 M NaCl. This fraction possessed both relaxing and supercoiling activities, and it was able to assemble regularly spaced nucleosomes onto naked DNA in an ATP‐dependent manner. Second, a negatively charged resin (heparin) was used, which retained small amounts of NAP‐2 (a very acidic polypeptide) and topoisomerase I. This fraction, although able to supercoil relaxed DNA, did so to a lesser extent than the Q‐Sepharose fraction. The data suggest that NAP‐2 is in complex(es) with other proteins, which are distinct from histones. © 2004 Wiley‐Liss, Inc.