Purification of annexin I and annexin II from human placental membranes by high-performance liquid chromatography

Protein Expr Purif. 1992 Feb;3(1):1-7. doi: 10.1016/1046-5928(92)90049-3.


Annexin I and annexin II were extracted from human placental membranes with ethylene glycol bis(beta-amino-ethyl ether)-N,N'-tetraacetic acid (EGTA) and purified by high-performance liquid chromatography by measuring their ability to inhibit phospholipase A2 activity in vitro. Neither protein was capable of binding to a DEAE-5PW HPLC column at neutral pH; however, they were resolved through binding to a Mono S column and passage through size-exclusion HPLC columns. Annexin I and its covalently linked dimer (36 and 66 kDa, respectively, by sodium dodecyl sulfate (SDS)-gel electrophoresis) reacted in one-dimensional immunoblots with monoclonal antibodies to annexin I and calpactin II, and with monoclonal and polyclonal antibodies to lipocortin I, confirming that annexin I, calpactin II, and lipocortin I are the same or closely related proteins. Milligram amounts of monomeric annexin I containing negligible amounts of the cross-linked dimeric annexin I were selectively isolated from placental membranes by using buffers containing the sulfhydryl reagent iodoacetic acid. Milligram amounts of cross-linked annexin I were selectively isolated when placental membranes were initially treated with buffers that did not contain iodoacetic acid and then extracted with Triton X-100, suggesting that sulfhydryl-dependent transglutaminase activity contributes to the selective isolation of this protein. A third phospholipase A2-inhibitory protein (35 kDa by SDS-gel electrophoresis) that reacted in immunoblots with monoclonal antibodies to calpactin I and annexin II, indicating their similar identity, was isolated. The procedure employed allows the rapid purification of annexins I and II in milligram amounts from placental membranes within 2 days.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Annexin A1 / isolation & purification*
  • Annexin A2 / isolation & purification*
  • Cell Membrane / chemistry
  • Chromatography, High Pressure Liquid*
  • Egtazic Acid
  • Female
  • Humans
  • Immunoblotting
  • Placenta / chemistry*
  • Pregnancy
  • Pregnancy Proteins / isolation & purification*


  • Annexin A1
  • Annexin A2
  • Pregnancy Proteins
  • Egtazic Acid