Purification of phospholipase C by hydrophobic interaction affinity chromatography

Abstract A simple procedure is described for the purification of phosphatidylcholine-hydrolyzing phospholipase C(PLC). Lecithin, the substrate for PLC, was ligated hydrophobically to octyl-Sepharose in 2 M (NH 4 ) 2 SO 4 . The washed lecithin-conjugated resin was then used to purify PLC from crude preparations by affinity chromatography. PLC binds to the lecithin moiety in the presence of Zn 2+ and is eluted with an acidic buffer containing EDTA. PLC activity was recovered in the eluate. Both sodium dodecyl sulphate polyacrylamide gel electrophoresis and p I electrofocusing showed that the eluate contained a single monomeric protein with an apparent molecular mass of 66 kDa and a p I of 5.5.